Department of Molecular Biology, University of Wyoming, P.O. Box 3944, Laramie, WY 82071-3944, USA
Received on March 16, 2002; revised on May 10, 2002; accepted on June 13, 2002
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Abstract |
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Key words: Drosophila/galactosyltransferase/insect/proteoglycan
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Introduction |
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It is established that humans encode at least seven ß4GalTs (Almeida et al., 1997, 1999; Lo et al., 1998
; Amado et al., 1999
; Okajima et al., 1999
). The members of this human gene family are designated ß4GalT-I to -VII, and each encodes an enzyme that transfers galactose from UDP-galactose in ß1,4 linkage to an acceptor substrate. The in vitro acceptor specificities of the human ß4GalTs have been defined, and the results indicate that these enzymes participate in glycoconjugate biosynthesis in vivo (Almeida et al., 1997
, 1999; Nomura et al., 1998
; Sato et al., 1998
; Schwientek et al., 1998
; Okajima et al., 1999
; Van Die et al., 1999
). However, the acceptor substrates recognized by different family members are not identical, because these enzymes can use N-acetylglucosamine, glucose, or xylose as acceptors. More specifically, the in vitro data indicate that ß4GalT-IVI are probably involved in glycoprotein and/or glycolipid biosynthesis, whereas ß4GalT-VII, also known as galactosyltransferase I, is involved in proteoglycan biosynthesis (Almeida et al., 1999
; Okajima et al., 1999
). The in vitro activity of ß4GalT-VII appears to reflect its true in vivo function because mammalian cell lines (Esko et al., 1987
), C. elegans (Herman and Horvitz, 1999
; Bulik et al., 2000
), and humans (Quentin et al., 1990
; Almeida et al., 1999
) with mutant ß4GalT-VII genes also have defects in glycosaminoglycan biosynthesis.
It was recently reported that Drosophila melanogaster encodes three putative ß4GalT genes (Altmann et al., 2001). However, no functional data are currently available to support the presumptive identification of these genes. In November 1999, we independently identified three putative ß4GalT genes in the Drosophila genome. This report focuses on one of these genes, CG11780 (Genbank Accession No. AE003750), because bioinformatic analyses predicted that this gene is an insect ortholog of human ß4GalT-VII and biochemical assays provided direct experimental evidence that the gene product is indeed a functional ß4GalT-VII. These findings, together with the recent identification of a Drosophila gene encoding a functional UDP-galactose transporter (Aumiller and Jarvis, 2002
; Segawa et al., 2002
), provide new information on the machinery available for glycosaminoglycan and proteoglycan biosynthesis in D. melanogaster and perhaps in other insects as well.
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Results |
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When the amino acid sequence encoded by CG11780 was used as the query for a second BLAST-P search, it was found to be most closely related to the seventh member of the human ß4GalT family and to the ß4GalT-VII encoded by the C. elegans sqv-3 gene (Table I). The putative Drosophila ß4GalT was nearly 50% identical to these proteins, as compared to about 30% identical to the other human ß4GalTs. In addition, the e values and bit scores associated with the human and C. elegans ß4GalT-VII proteins were significantly better than those obtained with any of the other human ß4GalTs. The phylogenetic tree analysis (Felsenstein, 1989) shown in Figure 1 also reveals that the putative Drosophila ß4GalT protein is more closely related to the known ß4GalT-VII proteins than to any of the other human ß4GalT family members.
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We also performed assays designed to determine if Dmß4GalT-VII had any in vitro activity toward several other acceptor substrates. Sf9 cells were transfected with the immediate early expression plasmid encoding Dmß4GalT-VII or the empty vector, as a negative control. Extracts were prepared and normalized amounts used for galactosyltransferase assays with various acceptor substrates. Assays were performed using boiled lysates or no acceptor to establish background for each assay, as described. The results of these experiments demonstrated that the expression plasmid encoding Dmß4GalT-VII only induced high levels of in vitro galactosyltransferase activity toward pNP-xylose (Figure 5A). By expanding the ordinates 10-fold, one can see evidence of minor activity toward pNP-GlcNAc (Figure 5C). However, the same result was obtained when Sf9 cells were transfected with the empty vector, suggesting that this activity was induced by transfection, independently of Dmß4GalT-VII expression. No significant activities were observed with pNP-GalNAc (Figure 5B) or glucocerebroside (Figure 5D) as the acceptor substrates. The ability of this Drosophila gene to induce high levels of activity toward ß-xylosyl but no other acceptor substrate tested strongly supports the identification of the gene product as a ß4GalT-VII.
Although it had been reported that 40 mM MnCl2 provides optimal human ß4GalT-VII activity (Almeida et al., 1999), as mentioned, it was of interest to examine the influence of various metals on the Drosophila enzyme. Galactosyltransferase assays were performed in the absence or presence of various metals using normalized extracts of Sf9 cells transfected with the immediate early expression plasmid encoding Dmß4GalT-VII (Figure 6). The results showed that in vitro transfer of galactose to pNP-xylose was strongly activated by manganese, slightly activated by cobalt, and very slightly activated by magnesium and nickel. No activity was observed in the absence of metals or in the presence of calcium or zinc. The influence of various concentrations of manganese and cobalt is shown in Figure 7. The optimal concentration of MnCl2 was 20 mM, which is half the optimal concentration for the human enzyme (Almeida et al., 1999
). The insect enzyme was also activated to a similar extent by either 20 or 40 mM CoCl2, but the highest activity observed in the presence of cobalt was still about 20 times less than that observed in the presence of manganese.
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Discussion |
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One way to obtain further information about insect cell glycan processing machinery is to use bioinformatics to search the Drosophila databases for genes encoding relevant functions. The emerging view that insect cells can produce galactosylated N-glycoproteins prompted us to use this approach to search for insect ß4GalT genes. Our search revealed that D. melanogaster encodes at least three putative ß4GalT genes. Each had previously been identified as such in Flybase (1999), solely on the basis of sequence similarity, and are probably the same as those previously reported by another group interested in insect glycobiology (Altmann et al., 2001
). However, when we expressed these genes and performed preliminary biochemical activity assays, we only obtained evidence of ß4GalT activity with CG11780 (Genbank Accession No. AE003750). Therefore, we focused the remainder of this study on this one gene, which appeared to be a Drosophila ortholog of human ß4GalT-VII.
An in vitro galactosyltransferase assay was used to examine endogenous ß4GalT activities in a Drosophila cell line and to determine the actual function of the CG11780 gene product. The results showed that Drosophila S2 cells have an enzymatic activity that can transfer galactose from UDP-galactose to ß-xylosyl but not to other acceptors in vitro. They also showed that this activity can be induced by transfecting Sf9 cells with an immediate early expression plasmid encoding the Drosophila CG11789 gene product. This enzyme had apparent Km values of about 50 µM and 1.2 mM for UDP-galactose and pNP-xylose, respectively; was strongly stimulated by MnCl2; and had an optimal pH of about 6.5. Finally, a GFP-tagged version of the gene product was localized to the Golgi apparatus of insect cells. The combined evidence obtained from the in silico, in vitro, and in vivo experiments reported in this manuscript indicates that the CG11780 gene does indeed encode a Drosophila cognate of human ß4GalT-VII. We designated this gene and its product Dmß4GalT-VII to conform to the nomenclature introduced by Lo and co-workers (1998) and concluded that it probably contributes to Drosophila proteoglycan biosynthesis by transferring galactose to the xylose residue in the linkage tetrasaccharide of glycosaminoglycan side chains.
The value of Drosophila as a model system for studying the role of glycosaminoglycans in proteoglycan function is well established. First, it is clear that this animal produces glycosaminoglycans and proteoglycans (Seppo and Tiemeyer, 2000; Toyoda et al., 2000b
; Selleck, 2001
). Second, it is clear that mutant phenotypes can arise from mutations in fly genes that appear to be involved in glycosaminoglycan biosynthesis. These genes include sugarless, sulfateless, and tout velu, which appear to encode Drosophila cognates of UDP-glucose dehydrogenase, N-deacetylase N-sulfotransferase, and heparan sulfate copolymerase, respectively (reviewed by Seppo and Tiemeyer, 2000
; Selleck, 2001
). The functional identification of these genes has been supported by direct structural analyses demonstrating that flies with mutations in these genes have the expected defects in glycosaminoglycan biosynthesis (Toyoda et al., 2000a
,b). However, to our knowledge, this report is the first to identify a Drosophila galactosyltransferase that is likely to be involved in glycosaminoglycan biosynthesis. According to Flybase (1999)
, this gene is located on chromosome 3R and has been cytologically mapped to 96B13-14. There are no recorded mutant alleles, but there is a deficiency (DF[3R]96B) in this region (96A2;96C2) that has a recessive lethal phenotype. Thus, future studies of Drosophila mutants could provide further information on the in vivo role of the Dmß4GalT-VII gene identified in this study and on the biological significance of proteoglycan production in this organism.
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Materials and methods |
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Plasmid constructs
The immediate early expression plasmid pAcP(-)IEdGalT3 was used to express the full-length Dmß4GalT-VII gene under the control of a baculovirus ie1 promoter. This plasmid was constructed by subcloning the 1.13 kb HincIIBamHI fragment from the Drosophila EST clone designated CK02622 (GenBank accession number AA142310; Research Genetics, Huntsville, AL; Rubin et al., 2000) into the SmaI and BglII sites of pAcP(-)IETV6 (Jarvis et al., 1996
). The expression plasmid pAc5.1DmGalT-VII-GFP was used to express a nearly full-length, C-terminally GFP-tagged version of the Dmß4GalT-VII protein under the transcriptional control of a Drosophila actin promoter. The 1.05 kb KpnIBspEI fragment, which encodes all but the last 12 amino acids of the Dmß4GalT-VII protein, was excised from CK02622, and the BspEI end was repaired with T4 DNA polymerase I. The resulting fragment was gel-purified and subcloned into the XmaI site of pEGFP-N1 (Clonetech Laboratories, Palo Alto, CA), which also had been repaired with DNA polymerase I. The resulting plasmid was isolated, amplified, purified, and used to excise the 1.82 kb KpnI/XbaI fragment encoding the Dmß4GalT-VII-GST fusion protein, which was gel-purified and subcloned into the corresponding sites of pAc5.1/A (Invitrogen). To produce each of these constructs, Escherichia coli transformants were isolated and screened using standard alkaline lysis plasmid isolation and restriction mapping procedures (Birnboim and Doly, 1979
; Sambrook et al., 1989
). The final plasmid preparations used for transient expression in insect cells were isolated from 200 ml transformed E. coli cultures by alkaline lysis (Birnboim and Doly, 1979
) and isopycnic ultracentrifugation on CsClethidium bromide density gradients (Sambrook et al., 1989
).
Transfection and extraction of Sf9 cells for biochemical assays
Sf9 cells were transfected with the purified immediate early expression plasmids described or the parental vectors, as negative controls, using a modified calcium phosphate method. Briefly, 2 x 106 cells were seeded into 25-cm2 tissue culture flasks (Corning, Corning, NY), washed with Graces medium, and transfected with 10 µg pAcIE1 plus 10 µg of the relevant expression plasmid or parental vector, as described previously (Jarvis et al., 1996). The cells were incubated for 2 h at 28°C, washed twice with complete TNM-FH, and fed with 5 ml of the same medium. The cells were then incubated for another 22 h at 28°C, removed from the culture flasks, washed with ice-cold buffer (25 mM TrisHCl, pH 7.4; 140 mM NaCl), and extracted with an appropriate galactosyltransferase buffer. The buffers used for each type of assay performed in this study were chosen from the literature to provide optimal activity in each type of ß4GalT assay. These buffers were: 25 mM sodium cacodylate, pH 7.0, containing 0.25% Triton-X-100 for pNP-xylose assays (Almeida et al., 1999
); 141 mM sodium cacodylate, pH 7.0, containing 0.7% Triton-X-100 and 10 mM ATP for pNP-GlcNAc and pNP-GalNAc assays (Bakker et al., 1994
); and 200 mM sodium cacodylate, pH 7.2, containing 0.375% Triton-X-100 for glucocerebroside assays (Nomura et al., 1998
). The cells were extracted for 10 min on ice, then the extracts were clarified by centrifugation for 10 min at 4°C at top speed in a microcentrifuge (Hermle Model Z180M). The supernatants were harvested and used for the assays.
Galactosyltransferase assays
Total protein concentrations in the cell extracts were determined using a commercial bicinchoninic assay kit (Pierce, Rockford, IL). Triplicate samples of each extract containing 50 µg total protein were then assayed in a total reaction volume of 0.1 ml of the appropriate buffer, as described. Each assay also contained various concentrations of various metals (specified in the figure legends), 0.3 µCi UDP-galactose, [galactose-1-3H(N)] (Perkin-Elmer Life Sciences, Boston, MA; 9.1 Ci/mmol), and 1.0 mM of the appropriate acceptor substrate (Sigma, St. Louis, MO; also specified in the figure legends). The reactions were incubated for 1 h at 37°C, then terminated by adding 0.5 ml ice-cold 50 mM ethylenediamine tetra-acetic acid, pH 8.0. The spent reactions were applied to Sep-Pak C18 cartridges (Millipore, Bedford, MA), unincorporated radioactivity was eliminated by washing with water, and incorporated radioactivity was eluted by washing with methanol. The eluants were added to 5 ml Scintisafe Plus 50% scintillation cocktail (Fisher Scientific, New Lawn, NJ), and radioactivity was measured in a Model LS-6500 liquid scintillation spectrometer (Beckman Coulter Instruments, Palo Alto, CA). All data were presented as the average pmol of galactose transferred per mg of total protein per h, with standard deviations calculated from the individual measurements.
The galactosyltransferase assay described was modified slightly to determine the apparent Km values for UDP-galactose and pNP-xylose. In each case, duplicate reactions were performed using 50 µg total protein from Dmß4GalT-VII-transfected Sf9 cell extracts prepared in pNP-xylose assay buffer supplemented with 20 mM MnCl2. To determine the apparent Km for the donor substrate, reactions were performed in the presence of 2.0 mM pNP-xylose, nonradioactive UDP-galactose concentrations ranging from 10500 µM, and a constant ratio of UDP-[galactose-1-3H(N)] with a specific activity of 9.1 Ci/mmol. To determine the apparent Km for the acceptor substrate, reactions were performed in the presence of 50 µM nonradioactive UDP-galactose, 0.3 µCi of UDP-[galactose-1-3H(N)], and pNP-xylose concentrations ranging from 0.15.0 mM.
Living cell fluorescence experiments
S2 cells were seeded into 25-cm2 tissue culture flasks (Corning) at a density of 3 x 106 cells, the cells were incubated overnight at 28°C, then they were transfected with 20 µg of either pAc5.1DmGalT-VII-GFP or pAc5.1/A using a standard calcium phosphate method described for use with the Drosophila Expression System (Invitrogen). The transfected cells were incubated with the DNA precipitate for 24 h at 28°C, washed, fed, and incubated for another 40 h at 28°C. The growth medium was then removed and the cells were stained for 1 h with fresh growth medium containing 350 nM BODIPY®TR ceramide (Molecular Probes, Eugene, OR). After staining, the cells were rinsed with fresh medium and examined under a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed; E-mail: DLJarvis@uwyo.edu
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References |
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Almeida, R., Levery, S.B., Mandel, U., Kresse, H., Schwientek, T., Bennett, E.P., and Clausen, H. (1999) Cloning and expression of a proteoglycan UDP-galactose:beta-xylose beta1, 4-galactosyltransferase I. A seventh member of the human beta4- galactosyltransferase gene family. J. Biol. Chem., 274, 2616526171.
Altmann, F., Fabini, G., Ahorn, H., and Wilson, I.B. (2001) Genetic model organisms in the study of N-glycans. Biochimie, 83, 703712.[CrossRef][ISI][Medline]
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nuc. Acids Res., 25, 33893402.
Amado, M., Almeida, R., Schwientek, T., and Clausen, H. (1999) Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. Biochim. Biophys. Acta, 1473, 3553.[ISI][Medline]
Aumiller, J.J. and Jarvis, D.L. (2002) Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems. Prot. Expr. Pur., forthcoming.
Bakker, H., Agterberg, M., Van Tetering, A., Koeleman, C.A., Van den Eijnden, D.H., and Van Die, I. (1994) A Lymnaea stagnalis gene, with sequence similarity to that of mammalian beta 14-galactosyltransferases, encodes a novel UDP-GlcNAc:GlcNAc beta-R beta 1
4-N-acetylglucosaminyltransferase. J. Biol. Chem., 269, 3032630333.
Bakker, H., Schoenmakers, P.S., Koeleman, C.A., Joziasse, D.H., Van Die, I., and Van den Eijnden, D.H. (1997) The substrate specificity of the snail Lymnaea stagnalis UDP-GlcNAc:GlcNAc beta-R beta 4-N-acetylglucosaminyltransferase reveals a novel variant pathway of complex-type oligosaccharide synthesis. Glycobiology, 7, 539548.[Abstract]
Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nuc. Acids Res., 7, 15131523.[Abstract]
Bulik, D.A., Wei, G., Toyoda, H., Kinoshita-Toyoda, A., Waldrip, W.R., Esko, J.D., Robbins, P.W., and Selleck, S.B. (2000) sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis. Proc. Natl Acad. Sci. USA, 97, 1083810843.
Esko, J.D., Weinke, J.L., Taylor, W.H., Ekborg, G., Roden, L., Anantharamaiah, G., and Gawish, A. (1987) Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I. J. Biol. Chem., 262, 1218912195.
Felsenstein, J. (1989) PHYLIPphylogeny inference package (version 3.2). Cladistics, 5, 164166.
Flybase. (1999) The Flybase database of the Drosophila genome projects and community literature. Nuc. Acids Res., 27, 8588. Available online at http://flybase.bio.indiana.edu.
Herman, T. and Horvitz, H.R. (1999) Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway. Proc. Natl Acad. Sci. USA, 96, 974979.
Hsu, T.A., Takahashi, N., Tsukamoto, Y., Kato, K., Shimada, I., Masuda, K., Whiteley, E.M., Fan, J.Q., Lee, Y.C., and Betenbaugh, M.J. (1997) Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells. J. Biol. Chem., 272, 90629070.
Jarvis, D.L., Weinkauf, C., and Guarino, L.A. (1996) Immediate early baculovirus vectors for foreign gene expression in transformed or infected insect cells. Prot. Expr. Pur., 8, 191203.[CrossRef][ISI]
Lo, N.W., Shaper, J.H., Pevsner, J., and Shaper, N.L. (1998) The expanding beta 4-galactosyltransferase gene family: messages from the databanks. Glycobiology, 8, 517526.
Nomura, T., Takizawa, M., Aoki, J., Arai, H., Inoue, K., Wakisaka, E., Yoshizuka, N., Imokawa, G., Dohmae, N., Takio, K., and others. (1998) Purification, cDNA cloning, and expression of UDP-Gal: glucosylceramide beta-1, 4-galactosyltransferase from rat brain. J. Biol. Chem., 273, 1357013577.
Ogonah, O.W., Freedman, R.B., Jenkins, N., Patel, K., and Rooney, B. (1996) Isolation and characterization of an insect cell line able to perform complex N-linked glycosylation on recombinant proteins. Bio/Technology, 14, 197202.[ISI]
Okajima, T., Yoshida, K., Kondo, T., and Furukawa, K. (1999) Human homolog of Caenorhabditis elegans sqv-3 gene is galactosyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. J. Biol. Chem., 274, 2291522918.
Quentin, E., Gladen, A., Roden, L., and Kresse, H. (1990) A genetic defect in the biosynthesis of dermatan sulfate proteoglycan: galactosyltransferase I deficiency in fibroblasts from a patient with a progeroid syndrome. Proc. Natl Acad. Sci. USA, 87, 13421346.[Abstract]
Rubin, G.M., Yandell, M.D., Wortman, J.R., Gabor-Miklos, G.L. Nelson, C.R., Hariharan, I.K., Fortini, M.E., Li, P.W., Apweiler, R., Fleischmann, W., and others. (2000) Comparative genomics of the eukaryotes. Science, 287, 22042215.
Rudd, P.M., Downing, A.K., Cadene, M., Harvey, D.J., Wormald, M.R., Weir, I., Dwek, R.A., Rifkin, D.B., and Gleizes, P.E. (2000) Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells. Biochemistry, 39, 15961603.[CrossRef][ISI][Medline]
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, New York.
Sato, T., Furukawa, K., Bakker, H., Van den Eijnden, D.H., and Van Die, I. (1998) Molecular cloning of a human cDNA encoding beta-1, 4- galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc beta-1, 4-galactosyltransferase. Proc. Natl Acad. Sci. USA, 95, 472477.
Schneider, I. (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morph., 27, 353365.[ISI][Medline]
Schwientek, T., Almeida, R., Levery, S.B., Holmes, E.H., Bennett, E., and Clausen, H. (1998) Cloning of a novel member of the UDP-galactose:beta-N-acetylglucosamine beta1, 4-galactosyltransferase family, beta4Gal-T4, involved in glycosphingolipid biosynthesis. J. Biol. Chem., 273, 2933129340.
Segawa, H., Kawakita, M., and Ishida, N. (2002) Human and Drosophila UDP-galactose transporters transport UDP-N-acetylgalactosamine in addition to UDP-galactose. Eur. J. Biochem., 269, 128138.
Selleck, S.B. (2001) Genetic dissection of proteoglycan function in Drosophila and C. elegans. Sem. Cell. Dev. Biol., 12, 127134.[CrossRef][ISI][Medline]
Seppo, A. and Tiemeyer, M. (2000) Function and structure of Drosophila glycans. Glycobiology, 10, 751760.
Sugumaran, G. and Silbert, J.E. (1991) Subfractionation of chick embryo epiphyseal cartilage Golgi. Localization of enzymes involved in the synthesis of the polysaccharide portion of proteochondroitin sulfate. J. Biol. Chem., 266, 95659569.
Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nuc. Acids Res., 22, 46734680.[Abstract]
Toyoda, H., Kinoshita-Toyoda, A., Fox, B., and Selleck, S.B. (2000a) Structural analysis of glycosaminoglycans in animals bearing mutations in sugarless, sulfateless, and tout-velu. Drosophila homologues of vertebrate genes encoding glycosaminoglycan biosynthetic enzymes. J. Biol. Chem. 275, 2185621861.
Toyoda, H., Kinoshita-Toyoda, A., and Selleck, S.B. (2000b) Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo. J. Biol. Chem., 275, 22692275.
Van Die, I., Van Tetering, A., Bakker, H., Van den Eijnden, D.H., and Joziasse, D.H. (1996) Glycosylation in lepidopteran insect cells: identification of a ß1, 4-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains. Glycobiology, 6, 157164.[Abstract]
Van Die, I., Van Tetering, A., Schiphorst, W.E., Sato, T., Furukawa, K., and Van den Eijnden, D.H. (1999) The acceptor substrate specificity of human beta4-galactosyltransferase V indicates its potential function in O-glycosylation. FEBS Lett., 450, 5256.[CrossRef][ISI][Medline]
Vaughn, J.L., Goodwin, R.H., Thompkins, G.J., and McCawley, P. (1977) The establishment of two insect cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro, 13, 213217.[ISI][Medline]