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• W1976493080 abstract "CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12–24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes. CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12–24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes. Sialic acids are a diverse family of negatively charged nine carbon 2-keto-3-deoxy sugars located at the termini of glycoproteins and glycolipids (1Schauer R. Kelm S. Reuter G. Roggentin P. Shaw L. Rosenberg A. Biology of the Sialic Acids. Plenum Press, New York1995: 7-67Crossref Google Scholar). More than 50 different types of naturally occurring sialic acids have been identified, with the most abundant sialic acid being N-acetylneuraminic acid (Neu5Ac). 2The abbreviations used are: Neu5Ac, N-acetylneuraminic acid; CSAS, CMP-sialic acid synthase; NLS, nuclear localization signal(s); RT, reverse transcription; GFP, green fluorescent protein; eGFP, enhanced GFP; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; FBS, fetal bovine serum; HPAEC, high performance anion exchange chromatography; CNS, central nervous system; HsNDmC, chimeric D. melanogaster CSAS containing a human leader; DmNHsC, chimeric human CSAS containing Drosophila leader.2The abbreviations used are: Neu5Ac, N-acetylneuraminic acid; CSAS, CMP-sialic acid synthase; NLS, nuclear localization signal(s); RT, reverse transcription; GFP, green fluorescent protein; eGFP, enhanced GFP; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; FBS, fetal bovine serum; HPAEC, high performance anion exchange chromatography; CNS, central nervous system; HsNDmC, chimeric D. melanogaster CSAS containing a human leader; DmNHsC, chimeric human CSAS containing Drosophila leader. Sialic acid can be present as a monomer or in a polymeric form at the termini of glycoconjugates and can be attached to acceptors in either an α(2,6), α(2,3), or α(2,8) linkage, which is determined by the specificity of different sialyltransferases (1Schauer R. Kelm S. Reuter G. Roggentin P. Shaw L. Rosenberg A. Biology of the Sialic Acids. Plenum Press, New York1995: 7-67Crossref Google Scholar). In mammals, the presence of sialic acid on proteins regulates their circulatory half-life, and sialic acids serve as ligands for endogenous lectins of the inflammatory and immune responses (2Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4951) Google Scholar). The sialylation pattern of lipids and proteins displayed on the cell surface regulates cell-cell interactions in normal development, and altered patterns are associated with tumorigenesis and oncogenic transformations in vertebrates (1Schauer R. Kelm S. Reuter G. Roggentin P. Shaw L. Rosenberg A. Biology of the Sialic Acids. Plenum Press, New York1995: 7-67Crossref Google Scholar, 3Fukuda M. Cancer Res. 1996; 56: 2237-2244PubMed Google Scholar, 4Takano R. Muchmore E. Dennis J.W. Glycobiology. 1994; 4: 665-674Crossref PubMed Scopus (74) Google Scholar). The regulated presence or absence of polysialic acid polymers on the neural cell adhesion molecule is required for proper establishment of the vertebrate embryonic nervous system (5Cunningham B.A. Hoffman S. Rutishauser U. Hemperly J.J. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3116-3120Crossref PubMed Scopus (152) Google Scholar, 6Hoffman S. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5762-5766Crossref PubMed Scopus (515) Google Scholar) and is associated with changes in neuronal plasticity that occur during learning and memory (7Kiss J.Z. Muller D. Rev. Neurosci. 2001; 12: 297-310Crossref PubMed Scopus (138) Google Scholar). Expression of sialic acids is widespread in the deuterostome lineage (vertebrates, ascidians, and echinoderms). Furthermore, certain pathogenic and commensal bacteria, viruses, and fungi are found to contain sialic acid as well (8Corfield A.P. Schauer R. Sialic Acids: Chemistry, Metabolism and Function. Springer-Verlag, New York1982: 196-200Google Scholar). On the other hand, sialic acid has been more difficult to detect in the protostome lineage (annelids, arthropods, and mollusks) (8Corfield A.P. Schauer R. Sialic Acids: Chemistry, Metabolism and Function. Springer-Verlag, New York1982: 196-200Google Scholar, 9Warren L. Comparative Biochemistry and Physiology. 1963; 10: 153-171Crossref PubMed Scopus (117) Google Scholar). However, since sialic acid was first reported to be present in Drosophila melanogaster embryos using lectin histochemistry and immunostaining (10Roth J. Kempf A. Reuter G. Schauer R. Gehring W.J. Science. 1992; 256: 673-675Crossref PubMed Scopus (144) Google Scholar, 11D'Amico P. Jacobs J.R. Tissue Cell. 1995; 27: 23-30Crossref PubMed Scopus (34) Google Scholar), a number of other studies using biochemical and genomic approaches have supported the presence of sialic acids in insects. Sialic acid in both a α(2,6) and α(2,8) linkage was detected in the vacuoles of the Malpighian tubules of Philaenus spumarius (cicada) larvae using both histochemistry and gas-liquid chromatography-mass spectroscopy (12Malykh Y.N. Krisch B. Gerardy-Schahn R. Lapina E.B. Shaw L. Schauer R. Glycoconj. J. 1999; 16: 731-739Crossref PubMed Scopus (49) Google Scholar). Whereas insect glycoproteins typically terminate with Man or GlcNAc in paucimannose, oligomannose, or hybrid structures (13Marchal I. Jarvis D.L. Cacan R. Verbert A. Biol. Chem. 2001; 382: 151-159Crossref PubMed Scopus (151) Google Scholar, 14Seppo A. Tiemeyer M. Glycobiology. 2000; 10: 751-760Crossref PubMed Scopus (62) Google Scholar), two insect cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1) have been shown to produce more complex glycan structures on expressed recombinant proteins, and DpN1 was shown to produce some sialylated glycans as well (15Palomares L.A. Joosten C.E. Hughes P.R. Granados R.R. Shuler M.L. Biotechnol. Prog. 2003; 19: 185-192Crossref PubMed Scopus (52) Google Scholar). Additionally, Watanabe et al. (17Watanabe S. Kokuho T. Takahashi H. Takahashi M. Kubota T. Inumaru S. J. Biol. Chem. 2002; 277: 5090-5093Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) showed that when recombinant proteins were expressed in Trichoplusia ni (Tn-5B1-4) cells in the presence of a hexosaminidase inhibitor, some sialylated glycans were detected. Using the availability of the complete Drosophila genome (19Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. George R.A. Lewis S.E. Richards S. Ashburner M. Henderson S.N. Sutton G.G. Wortman J.R. Yandell M.D. Zhang Q. Chen L.X. Brandon R.C. Rogers Y.H. Blazej R.G. Champe M. Pfeiffer B.D. Wan K.H. Doyle C. Baxter E.G. Helt G. Nelson C.R. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4744) Google Scholar), genes encoding enzymes in the sialic acid pathway have been identified and cloned. Expression of a functional D. melanogaster sialic acid 9-phosphate synthase (16Kim K. Lawrence S.M. Park J. Pitts L. Vann W.F. Betenbaugh M.J. Palter K.B. Glycobiology. 2002; 12: 73-83Crossref PubMed Scopus (52) Google Scholar) and α(2,6)-sialyltransferase (18Koles K. Irvine K.D. Panin V.M. J. Biol. Chem. 2004; 279: 4346-4357Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) provided genomic evidence for the presence of the sialic acid pathway in insects. In all known biological systems, sialylation of both lipids and proteins requires the metabolic generation of the sugar nucleotide CMP-Neu5Ac by a CMP-sialic acid synthetase (CSAS). The CMP-Neu5Ac is then transferred to an acceptor oligosaccharide in the Golgi apparatus by sialyltransferases (20Kean E.L. Glycobiology. 1991; 1: 441-447Crossref PubMed Scopus (74) Google Scholar). In the present study, we have identified and characterized a single D. melanogaster ortholog (DmCSAS) of mammalian and bacterial CMP-sialic acid synthetases as further evidence for the presence of the sialylation pathway in insects. In contrast to the vertebrate CMP-sialic acid synthetases, which are comprised of an N-terminal catalytic domain and an additional carboxyl-terminal domain, DmCSAS includes only the catalytic domain, similar to some bacterial enzymes, such as that of Neisseria meningitidis. In this study, DmCSAS is shown to be functional both in vivo and in vitro by complementation of a mammalian cell line, LEC29.Lec32 (21Potvin B. Raju T.S. Stanley P. J. Biol. Chem. 1995; 270: 30415-30421Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), which lacks CMP-sialic acid synthetase activity. Whereas mammalian CMP-sialic acid synthetase localizes to the nucleus (20Kean E.L. Glycobiology. 1991; 1: 441-447Crossref PubMed Scopus (74) Google Scholar, 22Kean E.L. Munster-Kuhnel A.K. Gerardy-Schahn R. Biochim. Biophys. Acta. 2004; 1673: 56-65Crossref PubMed Scopus (48) Google Scholar), the D. melanogaster enzyme is found primarily in the Golgi apparatus. In accordance with their differing intracellular locations, the mammalian protein begins with an N-terminal sequence containing a potential nuclear localization signal (NLS), whereas the D. melanogaster protein begins with an N-terminal sequence rich in hydrophobic amino acids characteristic of a signal/anchoring sequence. The different compartmental locations observed for the mammalian and insect CMP-sialic acid synthetases represent, to our knowledge, the first example of functionally equivalent enzymes localizing to different compartments in different eukaryotes. The DmCSAS exhibits its highest levels of mRNA expression during 14–17 h of embryonic development, similar to that observed for the D. melanogaster sialyltransferase (18Koles K. Irvine K.D. Panin V.M. J. Biol. Chem. 2004; 279: 4346-4357Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This occurrence is consistent with the finding of Roth et al. (10Roth J. Kempf A. Reuter G. Schauer R. Gehring W.J. Science. 1992; 256: 673-675Crossref PubMed Scopus (144) Google Scholar) concerning the presence of sialic acid and polysialic acid in specific stages of Drosophila development. In contrast to the ubiquitous tissue distribution of human CMP-sialic acid synthetase (HsCSAS) (23Lawrence S.M. Huddleston K.A. Tomiya N. Nguyen N. Lee Y.C. Vann W.F. Coleman T.A. Betenbaugh M.J. Glycoconj. J. 2001; 18: 205-213Crossref PubMed Scopus (61) Google Scholar), expression of the D. melanogaster mRNA is observed predominantly in the head in adults. Gene Identification, Isolation of a cDNA Clone, and DNA Sequencing—A BLAST search was performed using the tBLASTn algorithm at NCBI with the amino acid sequence of either the bacterial CMP-sialic acid synthetase (NeuA) (GenBank™ accession number J05023) or the mouse CMP-sialic acid synthetase (MmCSAS) (GenBank™ accession number MMU6215) as the query sequence. Two genomic clones (AE003515 and AC015229) representing the same genomic sequence on chromosome 3L in band 76D8 had significant homology to the query sequence. In addition, a single corresponding cDNA, GenBank™ accession number RH14248, was obtained from the normalized head library. An alignment of the predicted amino acid sequence of the DmC-SAS with other CMP-sialic acid synthetases suggested that this cDNA was truncated at the 5′ end, since translation from the first ATG would yield a protein lacking a highly conserved and functionally indispensable part of the protein. A full-length coding region of DmCSAS was obtained by RT-PCR using a forward primer derived from the genomic sequence using the nearest upstream ATG from the first ATG in the original cDNA and a reverse strand primer derived from the end of the coding region of the original cDNA. The predicted amino acid sequence of the cDNA obtained by RT-PCR now included a region shared by all members of the CMP-sialic acid synthetase family as well as an N-terminal extension rich in hydrophobic amino acids, typical of secretory signal sequences. The forward primer (CSA4; all primers are given in Table 1) contained a BamHI site, a Kozak sequence (GCCATC), and sequence corresponding to the first eight codons of DmCSAS. The reverse strand primer (CSA6N) contained a HindIII site, two in frame stop codons, and sequence representing the last seven codons of DmCSAS. Total RNA prepared by the TRIzol method (Invitrogen) from D. melanogaster (Oregon R, P2) 14–17-h dechorionated embryos treated with amplification grade DNase I (Invitrogen) was used as the template. First strand cDNA synthesis was performed using 0.6 μg of template RNA with the 3′-SMART rapid amplification of cDNA ends kit (BD Biosciences Clontech, Palo Alto, CA). 2.5 μl (of the 110-μl reverse transcription reaction) was introduced into a 50-μl PCR and amplified with Taq Gold using an Applied Biosystems GeneAmp 2400 thermal cycler using the following cycle settings: 95 °C for 5 min; 35 cycles at 95 °C for 1 min, 55 °C for 1.5 min, and 72 °C for 2 min; 72 °C for 10 min; hold at 4 °C. PCR reagents were purchased from Applied Biosystems (Foster City, CA). The 776-bp product was subcloned into the baculovirus vector pBlueBac4.5 (Invitrogen). The DNA sequence of this construct, pBlueBac-DmCSAS4, was determined on both strands using BigDye terminators (PerkinElmer Life Sciences) by the Nucleic Acid/Protein Core Research Facility of the Children's Hospital of Philadelphia. The DNA sequence of the cDNA matched the genomic sequence, except it lacked three introns of 52, 56, and 55 bp.TABLE 1Primers used for PCR amplification of human and Drosophila CSASPrimersSequencesForward primers CSA45′-CACTGGATCCGCCATCATGATAAAACTGAAACCGGCACT CS15′-CACTAAGCTTGCATTAATTTTGGCCAGGGGA CSA105′-CACTAGATCTGCCATCATGATAAAACTGAAACCGGCCACT HuCS15′-CACTAGATCTGCCATCATGGACTCGCTGGAGAAGGGG HuCS45′-GAAAATTGTCTGAGCAAGCCCCCGCACCTGGCAGCCCTA CSA125′-GGCCGAGGTGTGGAGAACGATATTCATGCATTAATTTTGGCCReverse strand primers CSA6N5′-AGTGAAGCTTTCACTATAACTCGGTTTTTGTTTCACT CS55′-AGTGAAGCTTCCAATCTTGGCGTCTTGGCCT CSA95′-AGTGGTCGACGTAACTCGGTTTTTGTTTCACTACT HuCS25′-AGTGGAATTCGTTTTTGGCATGAATTATTAACTTTTTC CSA135′-CAGGTGCGGGGGCTTGCTCAGACAATTTTCCGTGCACCC HuCS35′-TGCATGAATATCGTTCTCCACACCTCGGCCCTGGCC CSA115′-AGTGAAGCTTTCATTACTTGTACAGCTCGTCCATGCC CSA145′-AGTGGTCGACGGCTCAGACAATTTTCCGTGCACCC Open table in a new tab Amplification of Staged Drosophila cDNA Libraries, RNA, and cDNA—Total RNA was isolated from staged Drosophila or S2 cells as described. PCR was performed with either 1 μl of the head or larvalearly pupal cDNA libraries (obtained from the Berkeley Drosophila Genome Project consortium), or RT-PCR was performed with staged total RNA as described (42Kawasaki E.S. Innis M.A. Gelfand D.H. Snisky J.J. White T.J. PCR Protocols. Academic Press, San Diego, CA1990: 21-27Crossref Google Scholar) using 0.6 μg of RNA and Moloney murine leukemia virus reverse transcriptase (New England Biolabs, Beverly, MA) in 20 μl, which was subsequently introduced into a 100-μl PCR. PCR was performed with the forward primer CS1 and reverse strand primer CS5 as described above, except that 31 cycles were performed. PCR of the Drosophila Rapid-Scan Expression panels (Origene, Gaithersburg, MD) was performed in 25-μl reactions using the same PCR conditions already described, except that 35 cycles were used. Each row of the panel contains different amounts of cDNA, from 1× (1 pg of first strand cDNA) to 1000× (1 ng of cDNA), and cDNA for each developmental stage and tissue has been normalized to yield equivalent amplification of a ribosomal protein, RP49. PCR in 48-well microtiter plates (panels) was performed using an Applied Systems GeneAmp 9600 thermal cycler. Construction of Enhanced Green Fluorescent Protein (eGFP)-tagged Human and D. melanogaster CSAS and Human and D. melanogaster CSAS with Swapped Leader Sequences—To determine the intracellular localization of CSAS proteins, both the D. melanogaster CSAS and the human CSAS (23Lawrence S.M. Huddleston K.A. Tomiya N. Nguyen N. Lee Y.C. Vann W.F. Coleman T.A. Betenbaugh M.J. Glycoconj. J. 2001; 18: 205-213Crossref PubMed Scopus (61) Google Scholar) cDNAs were subcloned in plasmid pEGFP-N2 (BD Biosciences Clontech) to create a CSAS fusion protein with the eGFP. The resultant plasmids are called DmCSAS-GFP and HsCSAS-GFP. The coding region of DmCSAS was amplified by PCR of pBlueBac-DmCSAS4, using the forward primer, CSA10, containing a BglII site, an artificial Kozak sequence, and eight codons of DmCSAS and the reverse strand primer, CSA9, containing the last eight codons of DmCSAS, but not the stop codon, and an artificial SalI site. An extra C was added before the SalI site to keep the correct reading frame with eGFP. Similarly, the HsCSAS coding region was amplified by PCR using the forward primer, HsCS1, containing an artificial BglII site, a Kozak sequence, and the first seven codons of HsCSAS and the reverse strand primer, HsCS2, containing the last nine codons of HsCSAS, excluding the stop codon, an extra C, and an artificial EcoRI site. To determine the intracellular location of DmCSAS in Spodoptera frugiperda, the CSAS-GFP fusion protein was shuttled from the pEGFP vector into the baculovirus vector, pBlueBac4.5 by PCR amplification using the forward primer CSA10 and the reverse primer, CSA11 containing the last seven codons of GFP, two stop codons, and an artificial HindIII site. The resultant plasmid is called pBlueBac-DmCSAS-GFP. In order to ascertain whether the N-terminal sequences of both DmCSAS and HsCSAS were responsible for their distinct intracellular targeting, constructs were made that swapped the leader sequences of each protein with the other. An inspection of an alignment of many CSAS proteins (Fig. 1B), showed that eukaryotic CSAS proteins are longer than bacterial CSAS proteins at their N terminus and that these sequences are either rich in basic amino acids (mammalian), characteristic of NLS, or rich in hydrophobic amino acids (insects), characteristic of secretory signal sequences. We assigned sequences upstream of the start of the Escherichia coli CSAS in the alignment as “leader” sequences; thus, HsCSAS has a 40-amino acid leader, and DmCSAS has a 27-amino acid leader. A construct expressing the human CSAS protein with the D. melanogaster leader, DNHsC-GFP, was obtained by separately amplifying by PCR the leader of DmC-SAS using DmCSAS-GFP and the forward primer CSA10 and the reverse strand primer CSA13 containing eight codons of DmCSAS preceding the fusion point and five codons of HsCSAS past the fusion point and the nonleader fragment of HsCSAS using HsCSAS-GFP and the forward primer HsCS4 containing five codons of DmCSAS preceding the fusion point and eight codons of HsCSAS past the fusion point and the reverse strand primer, HsCS2. The resultant 113-bp leader fragment of DmCSAS was mixed with the 1207 bp downstream of the leader fragment of HsCSAS, which contain 30 nucleotides of complementarity, and amplified for 12 cycles with the outside primers, CSA10 and HsCS2, and the fused fragment was cloned into the BglII/EcoRI site of pEGFP-N2. Similarly, a construct expressing the D. melanogaster CSAS protein with the human leader, HsNDC-GFP, was obtained by separately amplifying by PCR the leader of HsCSAS using HsCSAS-GFP, the forward primer HsCS1, and the reverse strand primer HsCS3 (containing seven codons of HsCSAS upstream of the fusion point and five codons of DmC-SAS past the fusion point) and the downstream of leader fragment of DmCSAS using DmCSAS-GFP, the forward primer CSA12 (containing five codons of HsCSAS preceding the fusion point and nine codons of DmCSAS past the fusion point), and the reverse strand primer CSA9. The resultant 151-bp human leader was mixed with the 688-bp DmC-SAS downstream of leader fragment, which contained 30 nucleotides of complementarity, and amplified for 12 cycles using the outside primers HsCS1 and CSA9, and the fused fragment was cloned into the BglII/SalI site of pEGFP-N2. All final constructs were sequenced.FIGURE 1A, DmCSAS cDNA sequence. The DNA (top row) and amino acid (bottom row) sequences are shown. Note the presence of nine consecutive hydrophobic residues in the N-terminal region (underlined), suggestive of a secretory signal sequence/anchoring domain. The truncated cDNA clone RH14248 starts at bp 36 and is predicted to encode a protein shorter by 61 N-terminal amino acids. B, comparison of the amino acid sequences of the DmCSAS, HsCSAS, MmCSAS, EcCSAS, and NmC-SAS CMP-sialic acid synthase proteins. The protein sequences of each were aligned using the Pile-up and Pretty box programs of GCG (Accelrys, San Diego, CA), which uses progressive pairwise alignments. Gaps introduced to maximize homology are shown as dots. Identical amino acids are represented by black boxes, and similar amino acids are represented by gray boxes. The DmCSAS shares 35.5% identity with the HsCSAS, 36.3% identity with the MmCSAS, and 30.4% identity with the EcCSAS. Note that the DmCSAS and NmCSAS are short (about 240 amino acids), whereas the EcCSAS and mammalian CSAS are long (about 430 amino acids). However, among distantly related CSAS proteins, only the first 240 amino acids are conserved. Accession numbers are as follows: CR625607 (HsCSAS), MMU6215 (MmCSAS), J05023 (EcCSAS), and NMM95053 (NmC-SAS). C, phylogenetic relationship between the catalytic domains of various CSAS proteins. Multiple sequence alignment was performed with ClustalX, and the phylogenetic tree was calculated using the neighbor-joining method. The scale indicates distance in number of mutations per site. D, Kyte and Doolittle (window size of 9 amino acids) hydropathy plots of DmCSAS (i), AgCSAS (ii), MmCSAS (iii), HsC-SAS (iv), and O. mykiss CSAS (OmCSAS)(v). The bars in i and ii indicate the position of the putative N-terminal signal peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 1A, DmCSAS cDNA sequence. The DNA (top row) and amino acid (bottom row) sequences are shown. Note the presence of nine consecutive hydrophobic residues in the N-terminal region (underlined), suggestive of a secretory signal sequence/anchoring domain. The truncated cDNA clone RH14248 starts at bp 36 and is predicted to encode a protein shorter by 61 N-terminal amino acids. B, comparison of the amino acid sequences of the DmCSAS, HsCSAS, MmCSAS, EcCSAS, and NmC-SAS CMP-sialic acid synthase proteins. The protein sequences of each were aligned using the Pile-up and Pretty box programs of GCG (Accelrys, San Diego, CA), which uses progressive pairwise alignments. Gaps introduced to maximize homology are shown as dots. Identical amino acids are represented by black boxes, and similar amino acids are represented by gray boxes. The DmCSAS shares 35.5% identity with the HsCSAS, 36.3% identity with the MmCSAS, and 30.4% identity with the EcCSAS. Note that the DmCSAS and NmCSAS are short (about 240 amino acids), whereas the EcCSAS and mammalian CSAS are long (about 430 amino acids). However, among distantly related CSAS proteins, only the first 240 amino acids are conserved. Accession numbers are as follows: CR625607 (HsCSAS), MMU6215 (MmCSAS), J05023 (EcCSAS), and NMM95053 (NmC-SAS). C, phylogenetic relationship between the catalytic domains of various CSAS proteins. Multiple sequence alignment was performed with ClustalX, and the phylogenetic tree was calculated using the neighbor-joining method. The scale indicates distance in number of mutations per site. D, Kyte and Doolittle (window size of 9 amino acids) hydropathy plots of DmCSAS (i), AgCSAS (ii), MmCSAS (iii), HsC-SAS (iv), and O. mykiss CSAS (OmCSAS)(v). The bars in i and ii indicate the position of the putative N-terminal signal peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell Culture—LEC29.Lec32 cells (21Potvin B. Raju T.S. Stanley P. J. Biol. Chem. 1995; 270: 30415-30421Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), which are deficient in CMP-Neu5Ac synthetase activity, were grown at 37 °C in a humidified atmosphere with 5% CO2 in α-MEM (Invitrogen) medium supplemented with DNA and RNA precursors and 10% fetal bovine serum (FBS). 1 × 106 cells were plated on each well of a 6-well plate. After 24 h, the cells were transfected with 4 μg of DNA using Lipofectamine 2000 (Invitrogen) reagent. The cells were harvested 36 h post-transfection. The cells were washed once with Ca2+-, Mg2+-free PBS (Invitrogen) and harvested in 300 μl of mammalian protein extraction reagent (Pierce) containing HALT protease inhibitor mixture (Pierce). COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS in conditions similar to LEC29.Lec32 cells. Spodoptera Sf-9 were grown in serum-free Sf900 medium (Invitrogen) at 27 °C. Western Blotting and Detection of DmCSAS—The total protein of the cell lysate extracted with mammalian protein extraction reagent was determined using the BCA assay kit (Pierce) with a 96-well plate reader (Molecular Devices, Sunnyvale, CA). 50 μg of prepared protein was separated on a 12% SDS-polyacrylamide gel. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 10% blotting grade nonfat dry milk (Bio-Rad) in Tris-buffered saline, Tween 20 (TBST). DmCSAS-GFP was detected using a polyclonal mouse-anti-GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibody (1:1000 dilution) and visualized using a polyclonal goat anti-mouse IgG conjugated with horseradish peroxidase (1:1000 dilution) (Santa Cruz Biotechnology) and chemiluminescence substrate (Pierce). Immunostaining for Protein Localization—Immunostaining of COS-7 cells expressing either DmCSAS-GFP or HsCSAS-GFP was performed using organelle-specific markers and viewed by confocal microscopy. The CMP-sialic acid synthetase localization was determined by the co-localization of the eGFP-tagged protein and the organelle markers. COS-7 cells were plated in 4.2-cm2 chamber slides (LabTek, Campbell, CA) at a density of 200,000 cells/well. The cells were transfected after 12 h with 1 μg of DNA using Lipofectamine 2000. After 24 h, the cells were washed with PBS. The cells were then fixed with 4% formalin (Richard-Allan Scientific, Kalamazoo, MI) in PBS. After 15 min, the cells were permeabilized with 0.05% Triton X-100 (Sigma) in PBS for 2 min. The cells were then washed once w" @default.
• W1976493080 created "2016-06-24" @default.
• W1976493080 creator A5014510444 @default.
• W1976493080 creator A5041958361 @default.
• W1976493080 creator A5044750614 @default.
• W1976493080 creator A5063473597 @default.
• W1976493080 creator A5073720554 @default.
• W1976493080 creator A5074784454 @default.
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• W1976493080 date "2006-06-01" @default.
• W1976493080 modified "2023-10-16" @default.
• W1976493080 title "Expression of a Functional Drosophila melanogaster CMP-sialic Acid Synthetase" @default.
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