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Noboru Tomiya,
Someet Narang, Jung Park,
Badarulhisam Abdul-Rahman,
One Choi,
Sundeep Singh,
Jun Hiratake,
Kanzo Sakata,
Michael J Betenbaugh,
Karen B Palter,
Yuan C Lee
[show abstract]
[hide abstract]
ABSTRACT: Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.
Journal of Biological Chemistry 08/2006; 281(28):19545-60. · 4.77 Impact Factor
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Noboru Tomiya,
Someet Narang, Jung Park,
Badarulhisam Abdul-Rahman,
One Choi,
Sundeep Singh,
Jun Hiratake,
Kanzo Sakata,
Michael J. Betenbaugh,
Karen B. Palter,
Yuan C. Lee
[show abstract]
[hide abstract]
ABSTRACT: Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a β-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been
identified. We report the purification of a β-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary
N-glycan substrate, it exhibited a 5-fold preference for removal of the β(1,2)-linked N-acetylglucosamine from the Manα(1,3) branch compared with the Manα(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic
analysis suggested that Sfhex is an ortholog of mammalian lysosomal β-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as
the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells,
a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate
staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are
cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this
study suggests that Sfhex may play a role as a processing β-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.
Journal of Biological Chemistry 07/2006; 281(28):19545-19560. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
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.
Journal of Biological Chemistry 07/2006; 281(23):15929-40. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
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.
Journal of Biological Chemistry 06/2006; 281(23):15929-15940. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: A novel recombinant baculovirus expression vector was used to produce His-tagged human transferrin in a transformed insect cell line (Tn5beta4GalT) that constitutively expresses a mammalian beta-1,4-galactosyltransferase. This virus encoded the His-tagged human transferrin protein in conventional fashion under the control of the very late polyhedrin promoter. In addition, to enhance the synthesis of galactosylated biantennary N-glycans, this virus encoded human beta-1,2- N-acetylglucosaminyltransferase II under the control of an immediate-early (ie1) promoter. Detailed analyses by MALDI-TOF MS, exoglycosidase digestion, and two-dimensional HPLC revealed that the N-glycans on the purified recombinant human transferrin produced by this virus-host system included four different fully galactosylated, biantennary, complex-type glycans. Thus, this study describes a novel baculovirus-host system, which can be used to produce a recombinant glycoprotein with fully galactosylated, biantennary N-glycans.
Glycobiology 02/2003; 13(1):23-34. · 3.58 Impact Factor
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ABSTRACT: Elevated levels of homocysteine is a risk factor for coronary artery disease. Polymorphic alleles in the MTHFR genes that cause recessively inherited increased homocysteine level can explain only a small proportion of the observed variation in homocysteine level. To investigate additional genetic influences, we examined environmental, familial, and genetic influences on serum homocysteine levels in 661 family members of 112 probands who underwent elective coronary arteriography. Maximum likelihood methods were used to fit several genetic and non-genetic models of inheritance to these data to determine if an unobserved Mendelian major gene could explain the familial homocysteine distribution. Adjustments for age, lifestyle (smoking and alcohol consumption), serum folate and vitamin B12, and the measured genotype effect of the MTHFR C677T mutation was carried out separately for males and females using multiple regression models for homocysteine, before and after log-transformation prior to this segregation analysis. After excluding the effects of mutations in the MTHFR genes, we found evidence of a major gene acting in a co-dominant manner. Estimated mean homocysteine levels for the three putative genotypes (LL, LH, and HH) were 8.0, 10.1, and 15.9 mol/l, respectively, with relative frequencies of 56.8%, 37.2%, and 6%, respectively. Our analysis suggested the presence of a co-dominantly expressed major gene, in addition to the effects of the MTHFR C677T mutation. The results of this study also indicated that multifactorial inheritance was supported more strongly than Mendelian inheritance alone. Our findings may have implications for attempts to identify new homocysteine susceptible genes.
Human Genetics 07/2002; 111(2):128-135. · 5.07 Impact Factor
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ABSTRACT: In this study, we report the first cloning and characterization of a N-acetylneuraminic acid phosphate synthase gene from Drosophila melanogaster, an insect in the protostome lineage. The gene is ubiquitously expressed at all stages of Drosophila development and in Schneider cells. Similar to the human homologue, the gene encodes an enzyme with dual substrate specificity that can use either N-acetylmannosamine 6-phosphate or mannose 6-phosphate to generate phosphorylated forms of both the sialic acids, N-acetylneuraminic acid and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, respectively, when expressed in either bacterial or baculoviral expression systems. The identification of a functional sialic acid synthase in Drosophila indicates that insects have the biosynthetic capability to produce sialic acids endogenously. Although sialylation is widely distributed in organisms of the deuterstome lineage, genetic evidence concerning the presence or absence of sialic acid metabolism in organisms of the protostome lineage has been lacking. Homology searches of the Drosophila genome identified putative orthologues of other genes required for sialylation of glycoconjugates.
Glycobiology 03/2002; 12(2):73-83. · 3.58 Impact Factor
