Papers by Edoardo Giacopuzzi
BMC Biochemistry, 2011
Background: Large surface loops contained within compact protein structures and not involved in c... more Background: 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 βsheets 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. Results: Gallus gallus (Gg) genome contains one NEU3 gene encoding a protein with a unique 188 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 80 amino acid loop characteristic of mammalian NEU4. 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 NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence.
PLoS ONE, 2014
The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes tha... more The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (p<0.05), namely c.650T>C and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.
Glycobiology, Jan 28, 2015
Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9... more Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase (Lse), is detected. Using the homology modeling approach we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum ...
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2014
The lysosomal hydrolase galactocerebrosidase (GALC) catalyzes the removal of galactose from galac... more The lysosomal hydrolase galactocerebrosidase (GALC) catalyzes the removal of galactose from galactosylceramide and from other sphingolipids. GALC deficiency is responsible for globoid cell leukodystrophy (GLD), or Krabbe's disease, an early lethal inherited neurodegenerative disorder characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system (CNS). The poor outcome of current clinical treatments calls for novel model systems to investigate the biological impact of GALC down-regulation and for the search of novel therapeutic strategies in GLD. Zebrafish (Danio rerio) represents an attractive vertebrate model for human diseases. Here, lysosomal GALC activity was demonstrated in the brain of zebrafish adults and embryos. Accordingly, we identified two GALC co-orthologs (named galca and galcb) dynamically co-expressed in CNS during zebrafish development. Both genes encode for lysosomal enzymes endowed with GALC activity. Single down-regulation of galca or galcb by specific antisense morpholino oligonucleotides results in a partial decrease of GALC activity in zebrafish embryos that was abrogated in double galca/galcb morphants. However, no psychosine accumulation was observed in galca/galcb double morphants. Nevertheless, double galca/galcb knockdown caused reduction and partial disorganization of the expression of the early neuronal marker neuroD and an increase of apoptotic events during CNS development. These observations provide new insights into the pathogenesis of GLD, indicating that GALC loss-of-function may have pathological consequences in developing CNS independent of psychosine accumulation. Also, they underscore the potentiality of the zebrafish system in studying the pathogenesis of lysosomal neurodegenerative diseases, including GLD.
Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutat... more Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene coding for mucolipin-1 (TRPML1). TRPML1 belongs to a transient receptor potential channels (TRP) subfamily, which in mammals includes two other members: mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3). Bioinformatic analysis of the Danio rerio (zebrafish) genome and trascriptome revealed the presence of five different genes related to human mucolipins: mcoln1.1, mcoln1.2, mcoln2, mcoln3.1 and mcoln3.2. We focused our efforts on the characterization of the two putative zebrafish MCOLN1 co-orthologs. Transient-expression experiments in human HeLa cells demonstrated that fish Mcoln1.1 and Mcoln1.2, similarly to TRPML1, localize to late endosomal/lysosomal compartments. Real-Time PCR (RT-PCR) experiments showed that both genes are maternally expressed and transcribed at different levels during embryogenesis. RT-PCR analysis in different zebrafish tissues displayed ubiquitary expression for mcoln1.1 and a more tissue-specific pattern for mcoln1.2. Spatial and temporal expression studies using whole-mount in situ hybridization confirmed that both genes are maternally expressed and ubiquitously transcribed during gastrulation and early somitogenesis. Notably, in the next developmental stages they are more expressed in neural regions and in retina layers, tissues affected in MLIV. Interestingly, mcoln1.1 is detected, from 10 somite-stage until to 36 hpf, in the yolk syncytial layer (YSL) and in the intermediate cell mass (ICM), the earliest site of hematopoiesis. Overall, the redundancy of mucolipins together with their expression profile support the biological relevance of this class of proteins in zebrafish. The data herein presented indicate that Danio rerio could be a suitable vertebrate model for the study of some aspects of MLIV pathogenesis.
The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes tha... more The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal
sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal
storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes:
sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total
of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the
NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human
population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI
GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making
it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and
cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate
variants tested, only two resulted in significantly lower levels of sialidase activity (p,0.05), namely c.650T.C and c.700G.A.
These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including
either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type
enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological
mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously
unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.
The aim of this study is to shed light on the functional role of slc7a6os, a gene highly conserve... more The aim of this study is to shed light on the functional role of slc7a6os, a gene highly conserved in vertebrates. The Danio rerio slc7a6os gene encodes a protein of 326 amino acids with 46% identity to human SLC7A6OS and 14% to Saccharomyces cerevisiae polypeptide Iwr1. Yeast Iwr1 specifically binds RNA pol II, interacts with the basal transcription machinery and regulates the transcription of specific genes. In this study we investigated for the first time the biological role of SLC7A6OS in vertebrates. Zebrafish slc7a6os is a maternal gene that is expressed throughout development, with a prevalent localization in the developing central nervous system (CNS). The gene is also expressed, although at different levels, in various tissues of the adult fish. To determine the functional role of slc7a6os during zebrafish development, we knocked-down the gene by injecting a splice-blocking morpholino. At 24 hpf morphants show morphological defects in the CNS, particularly the interface between hindbrain and midbrain is not well-defined. At 28 hpf the morpholino injected embryos present an altered somite morphology and appear partially or completely immotile. At this stage the midbrain, hindbrain and cerebellum are compromised and not well defined compared with control embryos. The observed alterations persist at later developmental stages. Consistently, the expression pattern of two markers specifically expressed in the developing CNS, pax2a and neurod, is significantly altered in morphants. The co-injection of embryos with synthetic slc7a6os mRNA, rescues the morphant phenotype and restores the wild type expression pattern of pax2a and neurod. Our data suggest that slc7a6os might play a critical role in defined areas of the developing CNS in vertebrates, probably by regulating the expression of key genes.
Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from... more Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.
OBJECTIVE:
The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesiv... more OBJECTIVE:
The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesive and angiogenic growth factor (AGF) proteins, inducing endothelial cell (EC) adhesion and proangiogenic activation. Tat was exploited to investigate the events triggered by EC adhesion to substrate-bound AGF that lead to proangiogenic activation.
METHODS AND RESULTS:
Immobilized Tat induces actin cytoskeleton organization, formation of α(v)β(3) integrin(+)focal adhesion plaques, and recruitment of vascular endothelial growth factor receptor-2 (VEGFR2) in the ventral plasma membrane of adherent ECs. Also, acceptor photobleaching fluorescence resonance energy transfer demonstrated that VEGFR2/α(v)β(3) coupling occurs at the basal aspect of Tat-adherent ECs. Cell membrane fractionation showed that a limited fraction of α(v)β(3) integrin and VEGFR2 does colocalize in lipid rafts at the basal aspect of Tat-adherent ECs. VEGFR2 undergoes phosphorylation and triggers pp60src/ERK(1/2) activation. The use of lipid raft disrupting agents and second messenger inhibitors demonstrated that intact lipid rafts and the VEGFR2/pp60src/ERK(1/2) pathway are both required for cytoskeleton organization and proangiogenic activation of Tat-adherent ECs.
CONCLUSIONS:
Substrate-immobilized Tat causes VEGFR2/α(v)β(3) complex formation and polarization at the basal aspect of adherent ECs, VEGFR2/pp60src/ERK(1/2) phosphorylation, cytoskeleton organization, and proangiogenic activation. These results provide novel insights in the AGF/tyrosine kinase receptor/integrin cross-talk.
Background
Large surface loops contained within compact protein structures and not involved in ca... more Background
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 beta-propeller composed by anti-parallel beta-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.
Results
Gallus gallus (Gg) genome contains one NEU3 gene encoding a protein with a unique 188 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 80 amino acid loop characteristic of mammalian NEU4. Based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved beta-strands of the fifth blade of the sialidase beta-propeller. Moreover this loop is highly variable in sequence and size in NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence.
Conclusion
In this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved beta-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 NEU3, this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution.
JAIDS Journal of …, Jan 1, 2011
Here we demonstrate that EC adhesion to substrate-immobilized Tat induces the recruitment in the ... more Here we demonstrate that EC adhesion to substrate-immobilized Tat induces the recruitment in the ventral plasma membrane (VPM) of αvβ3, paxillin, focal adhesion kinase (FAK), pp60src and the vascular endothelial growth factor receptor-2 (VEGFR2). As expected, paxillin ...
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Papers by Edoardo Giacopuzzi
sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal
storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes:
sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total
of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the
NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human
population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI
GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making
it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and
cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate
variants tested, only two resulted in significantly lower levels of sialidase activity (p,0.05), namely c.650T.C and c.700G.A.
These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including
either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type
enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological
mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously
unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.
The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesive and angiogenic growth factor (AGF) proteins, inducing endothelial cell (EC) adhesion and proangiogenic activation. Tat was exploited to investigate the events triggered by EC adhesion to substrate-bound AGF that lead to proangiogenic activation.
METHODS AND RESULTS:
Immobilized Tat induces actin cytoskeleton organization, formation of α(v)β(3) integrin(+)focal adhesion plaques, and recruitment of vascular endothelial growth factor receptor-2 (VEGFR2) in the ventral plasma membrane of adherent ECs. Also, acceptor photobleaching fluorescence resonance energy transfer demonstrated that VEGFR2/α(v)β(3) coupling occurs at the basal aspect of Tat-adherent ECs. Cell membrane fractionation showed that a limited fraction of α(v)β(3) integrin and VEGFR2 does colocalize in lipid rafts at the basal aspect of Tat-adherent ECs. VEGFR2 undergoes phosphorylation and triggers pp60src/ERK(1/2) activation. The use of lipid raft disrupting agents and second messenger inhibitors demonstrated that intact lipid rafts and the VEGFR2/pp60src/ERK(1/2) pathway are both required for cytoskeleton organization and proangiogenic activation of Tat-adherent ECs.
CONCLUSIONS:
Substrate-immobilized Tat causes VEGFR2/α(v)β(3) complex formation and polarization at the basal aspect of adherent ECs, VEGFR2/pp60src/ERK(1/2) phosphorylation, cytoskeleton organization, and proangiogenic activation. These results provide novel insights in the AGF/tyrosine kinase receptor/integrin cross-talk.
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 beta-propeller composed by anti-parallel beta-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.
Results
Gallus gallus (Gg) genome contains one NEU3 gene encoding a protein with a unique 188 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 80 amino acid loop characteristic of mammalian NEU4. Based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved beta-strands of the fifth blade of the sialidase beta-propeller. Moreover this loop is highly variable in sequence and size in NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence.
Conclusion
In this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved beta-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 NEU3, this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution.
sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal
storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes:
sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total
of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the
NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human
population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI
GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making
it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and
cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate
variants tested, only two resulted in significantly lower levels of sialidase activity (p,0.05), namely c.650T.C and c.700G.A.
These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including
either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type
enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological
mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously
unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.
The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesive and angiogenic growth factor (AGF) proteins, inducing endothelial cell (EC) adhesion and proangiogenic activation. Tat was exploited to investigate the events triggered by EC adhesion to substrate-bound AGF that lead to proangiogenic activation.
METHODS AND RESULTS:
Immobilized Tat induces actin cytoskeleton organization, formation of α(v)β(3) integrin(+)focal adhesion plaques, and recruitment of vascular endothelial growth factor receptor-2 (VEGFR2) in the ventral plasma membrane of adherent ECs. Also, acceptor photobleaching fluorescence resonance energy transfer demonstrated that VEGFR2/α(v)β(3) coupling occurs at the basal aspect of Tat-adherent ECs. Cell membrane fractionation showed that a limited fraction of α(v)β(3) integrin and VEGFR2 does colocalize in lipid rafts at the basal aspect of Tat-adherent ECs. VEGFR2 undergoes phosphorylation and triggers pp60src/ERK(1/2) activation. The use of lipid raft disrupting agents and second messenger inhibitors demonstrated that intact lipid rafts and the VEGFR2/pp60src/ERK(1/2) pathway are both required for cytoskeleton organization and proangiogenic activation of Tat-adherent ECs.
CONCLUSIONS:
Substrate-immobilized Tat causes VEGFR2/α(v)β(3) complex formation and polarization at the basal aspect of adherent ECs, VEGFR2/pp60src/ERK(1/2) phosphorylation, cytoskeleton organization, and proangiogenic activation. These results provide novel insights in the AGF/tyrosine kinase receptor/integrin cross-talk.
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 beta-propeller composed by anti-parallel beta-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.
Results
Gallus gallus (Gg) genome contains one NEU3 gene encoding a protein with a unique 188 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 80 amino acid loop characteristic of mammalian NEU4. Based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved beta-strands of the fifth blade of the sialidase beta-propeller. Moreover this loop is highly variable in sequence and size in NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence.
Conclusion
In this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved beta-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 NEU3, this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution.