Glycobiology, 2000, Vol. 10, No. 8 837-847
© 2000 Oxford University Press
N-glycan patterns of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase
Department of Chemical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA, 2GlycoLab, Central Research Institute, Nakano Vinegar Co. Ltd., 2–6 Nakamura-cho, Handa-City Aichi 475–8585, Japan, 3Suntory Institute for Bioorganic Research, Wakayama-dai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan, the 4Department of Microbiology, University of Wyoming, Laramie, WY 82071, USA, and 5Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
Received on December 8, 1999; revised on February 8, 2000; accepted on February 8, 2000.
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
|---|
The N-glycans of human serum transferrin produced in Trichopulsia ni cells were analyzed to examine N-linked oligosaccharide processing in insect cells. Metabolic radiolabeling of the intra- and extracellular protein fractions revealed the presence of multiple transferrin glycoforms with molecular weights lower than that observed for native human transferrin. Consequently, the N-glycan structures of transferrin in the culture medium were determined using three-dimensional high performance liquid chromatography. The attached oligosaccharides included high mannose, paucimannosidic, and hybrid structures with over 50% of these structures containing one fucose,
(1,6)-, or two fucoses, (1,6)- and (1,3)-, linked to the Asn-linked N-acetylglucosamine. Neither sialic acid nor galactose was detected on any of the N-glycans. However, when transferrin was coexpressed with ß(1,4)-galactosyltransferase three additional galactose-containing hybrid oligosaccharides were obtained. The galactose attachments were exclusive to the (1,3)-mannose branch and the structures varied by the presence of zero, one, or two attached fucose residues. Furthermore, the presence of the galactosyltransferase appeared to reduce the number of paucimannosidic structures, which suggests that galactose attachment inhibits the ability of hexosaminidase activity to remove the terminal N-acetylglucosamine. The ability to promote galactosylation and reduce paucimannosidic N-glycans suggests that the oligosaccharide processing pathway in insect cells may be manipulated to mimic more closely that of mammalian cells. Key words: baculovirus/glycosylation/glycosyltransferase/oligosaccharide/expression
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
|---|
The Baculovirus Expression Vector System (BEVS) has become increasingly popular for the expression of recombinant proteins (Luckow and Summers, 1988
; Luckow, 1995). One of the key factors that has contributed to the wide spread use of this system is its ability to produce relatively large quantities of posttranslationally modified eukaryotic proteins (O'Reilly et al., 1992; Klenk, 1996; Jarvis, 1997) often including peptide glycosylation. Unlike mammalian cells, however, the oligosaccharide processing pathway in insect cells is not well characterized (Marz et al., 1995; Altmann, 1996). Insects represent the majority of the known animal species (Kubelka et al., 1994) and are widely used in developmental and recombinant protein studies. Therefore, identifying and detailing the differences in oligosaccharide processing between mammalian and insect cells is of scientific interest. Furthermore, the ability to manipulate the oligosaccharide processing pathway of insect cells may be useful for exploiting the benefits of the baculovirus system across a wider spectrum of applications.
Experimental evidence suggests that glycoproteins produced in insect cells possess N-linked oligosaccharides that are principally comprised of high mannose and truncated low mannose (paucimannosidic) structures (Butters and Hughes, 1981; Hsieh and Robbins, 1984; Kuroda et al., 1990; Chen and Bahl, 1991; Kulakosky et al., 1998). Even though activities for ß(1,2)-N-acetylglucosaminyltransferase I (GlcNAcTI) (Altmann et al., 1993; Velardo et al., 1993), ß(1,2)-N-acetylglucosaminyltransferase II (GlcNAcTII) (Altmann et al., 1993), and ß(1,4)-galactosyltransferase (GalT) (van Die et al., 1996) have been detected in insect cells, N-glycan structures containing terminal N-acetylglucosamine (GlcNAc) and galactose (Gal) residues are not widely observed on native or heterologous glycoproteins. However, recent studies of both homologous (Hard et al., 1993; Kubelka et al., 1994) and heterologous (Davidson et al., 1990; Ogonah et al., 1996) proteins obtained from insect cell sources have revealed the presence of a limited number of hybrid and complex type N-glycan structures.
We previously reported on the variability of N-glycan structures that exist between the intracellular and secreted forms of a recombinant immunoglobulin G (IgG) (Hsu et al., 1997). The presence of GlcNAc at the terminal position of some N-glycans indicates that insect cells produce the acceptor substrate for galactosylation. However, the processing of the N-linked oligosaccharides of IgG is highly variable (Radmacher et al., 1986; Tandai et al., 1991) so a secreted protein with more defined carbohydrate structures was chosen as a candidate for further study. As a result, human serum transferrin (hTf) has been expressed and purified from insect cells for oligosaccharide analysis of the attached N-glycans.
Transferrin is a serum glycoprotein found in the physiological fluids of vertebrates (Aisen, 1989; Thorstensen and Romslo, 1990) and insect larva (Bartfeld and Law, 1990) that is responsible for carrying Fe+3 to all cells in the body. When bound to iron the circulating transferrin is recognized by a specific surface receptor on cells and internalized to release iron into the cytoplasm (Trowbridge et al., 1984). Other members of the 80 kDa glycoprotein family include lactoferrin, melanotransferrin, and ovotransferrin (Montreuil et al., 1997). Serum transferrin also plays a role in host defense by depriving any circulating microorganisms of essential iron (Bullen et al., 1990).
Substantial information exists about the carbohydrate structure of hTf and other transferrins. HTf has two potential N-linked glycosylation sites in its carboxy-terminal domain at Asn413 and Asn611 (MacGillivray et al., 1983). Previous studies have shown that the transferrin glycoforms present in human serum are comprised of species having terminally sialylated bi-, tri-, and, tetrantennary oligosaccharides (Leger et al., 1989; Fu and van Halbeek, 1992). The most dominant glycoform includes biantennary oligosaccharides located at both asparagine positions, although, changes in physiological conditions can affect the N-glycan pattern observed in the host (Montreuil et al., 1997). Very little is known about the N-glycan structures of hTf expressed in foreign hosts. Recombinant hTf expressed in baby hamster kidney cells was comprised of numerous glycoforms, however, the detailed N-glycan structures were not determined (Mason et al., 1993). In related studies, both human and bovine lactoferrins have been expressed in insect cells and subjected to glycosylation analysis. N-linked oligosaccharides obtained from bovine lactoferrin expressed in Mamestra brassicae cells were observed to lack any complex or hybrid structures (Lopez et al., 1997). Carbohydrate analysis revealed a small fraction of Gal in oligosaccharides obtained from N-glycans of human lactoferrin expressed in Spodoptera frugiperda (Sf-9) (Salmon et al., 1997), though the analysis was for monosaccharides only.
One possible reason for the limitation in N-glycan processing of glycoproteins in insect cells is a deficiency in the enzymes necessary to produce complex oligosaccharides. In a recent study, coexpression of a recombinant GlcNAcTI in Sf-9 cells resulted in an increase of terminal GlcNAc residues found on the N-glycans of fowl plague virus hemagglutinin (Wagner et al., 1996). Additional studies used lectin blot analysis to demonstrate that the expression of GalT in Sf-9 cells led to the incorporation of terminal ß-linked Gal residues in the N-glycans of baculovirus and heterologous glycoproteins (Jarvis and Finn, 1996; Hollister et al., 1998). To gain greater insight into the oligosaccharide processing pathway of insect cells, an analysis of the N-glycans from recombinant hTf was undertaken in the current study. In addition, the oligosaccharides generated following coexpression of GalT were detailed in order to elucidate the structural changes resulting from expression of heterologous oligosaccharide modifying enzymes.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
|---|
Expression of recombinant hTf in insect cells
The recombinant human transferrin baculovirus, AcNPV-hTf, was harvested 5 days following transfection of Sf-9 cells, plaque purified, and amplified to provide a working virus stock. To verify expression of hTf in insect cells, clarified cell lysates were prepared from T.ni cells infected 3 days with AcNPV-hTf or a negative control baculovirus (AcBB) (Lindsay et al., 1993) and subjected to western blot analysis. The immunoblot revealed a major immunoreactive band of ~78 kDa in cell lysates of cells that were infected with AcNPV-hTf (Figure 1).
|
T.ni cells were observed to produce higher levels of secreted hTf than Sf-9 cells and were consequently used for subsequent oligosaccharide analysis. Medium from T.ni cells infected 3 days with AcNPV-hTf was separated by SDS–PAGE and stained with Coomassie brilliant blue to examine extracellular hTf expression levels. Recombinant hTf is clearly visible in the medium (Figure 2). No band corresponding to hTf was observed in the medium from uninfected cells. The molecular weight (MW) of hTf produced in T.ni cells is slightly lower than its native counterpart.
|
Effects of tunicamycin on hTf produced in T.ni cells
To examine the contribution of N-linked oligosaccharides to the MW of recombinant hTf, T.ni cells were labeled 4 h with 35S-methionine after being infected 48 h with AcNPV-hTf in the presence or absence of tunicamycin. Following the labeling period, cells were lysed, subjected to SDS–PAGE, dried, and autoradiographed. Secreted hTf was immunoprecipitated from the medium using a polyclonal
-hTf antibody and similarly processed. The autoradiograph depicted in Figure 3 shows that the presence of tunicamycin, an inhibitor of asparagine-linked oligosaccharide processing, results in production of a lower MW form of intracellular and secreted hTf. In the absence of tunicamycin, at least three distinct hTf bands can be seen in the whole cell lysates (Figure 3A). The lower band (n) appears to be non-glycosylated hTf since it is identical in MW to the hTf produced in the presence of tunicamycin. The secreted hTf fraction also appears to contain multiple hTf glycoforms (Figure 3B) which collapse to a lower MW form in the presence of tunicamycin.
|
Analysis of secreted hTf produced in insect cells
To determine if altering the intracellular level of an enzyme in the oligosaccharide processing pathway can promote elongation of hTf N-glycans, a recombinant GalT was overexpressed in insect cells in conjunction with hTf.
Two T.ni insect cell cultures were infected with AcNPV-hTf. One of the cultures was also coinfected with the recombinant AcP(+)IE1GalT baculovirus, which contains the gene for GalT under the IE1 promoter. The medium from each of these cell cultures was collected at 66 h pi and hTf was purified on a phenyl Sepharose hydrophobic column. Pooled fractions (Figure 4) containing 
95% hTf were separated by SDS–PAGE, transferred to a PVDF membrane, and were analyzed for Gal content using HPAEC/PAD following TFA hydrolysis of the hTf bands (Weitzhandler et al., 1993; Fan et al., 1994). Analysis of the monosaccharides indicated that the hTf oligosaccharides from T.ni cells coinfected with the GalT recombinant baculovirus contained a measurable amount of Gal while the samples without GalT did not contain significant Gal levels (unpublished observations).
|
S ince the monosaccharide analysis indicated a difference in the Gal levels of the hTf N-glycans from cells in the presence or absence of GalT expression, a more detailed comparison of the N-glycans was undertaken using HPLC analysis. N-glycans were released from recombinant transferrin by glycoamidase A (sweet almond). Based on total carbohydrate analysis, more than 80% of the carbohydrate chains were released by digestion. The reducing ends of the released N-glycans were reductively aminated with 2-aminopyridine. No acidic oligosaccharides were detected on a DEAE-5PW column. The PA-oligosaccharides were then separated by a reversed phase column (Figure 5). Each separated fraction (A through L) was then applied to an amide column. Fraction B was further separated into two fractions B1 and B2 on the amide column while fractions E, F, G, and J were similarly separated into E1 and E2; F1, F2, and F3; G1 and G2; and J1, J2, and J3, respectively (unpublished observations). Three distinct peaks, G2, I, and K, were shown to exist only in the cells coinfected with the GalT baculovirus.
|
Structural characterization of PA-oligosaccharides
Structural assignment of all neutral N-glycans from recombinant transferrin was performed by a 2-D mapping technique as described previously (Takahashi et al., 1995
). The coordinates of almost all the N-glycans coincided (±5%) with those of known oligosaccharides on the map. PA-oligosaccharides A, B1, B2, C, D, E1, E2, F1, F2, F3, G1, G2, H, J1, J2, J3, K, and L were assigned as code numbers M8.1, M7.2, M9.1, M7.1, M6.1, M9.2, M.8.2, M5.1, 000.1, 100.2, M.6.10, 100.4, 010.1F, 010.0, 010.1, 110.2, 110.4, and 110.1, respectively (Table I). Co-chromatography of each of the sample PA-oligosaccharides with the corresponding reference compound on ODS and amide columns confirmed these assignments.
|
The structural candidate for glycan E1 was identified as M9.2, which has a Glc
3Man2Man2Man3Manß4 sequence. Although the presence of glucose is unusual, we confirmed the structure by co-chromatography with the reference oligosaccharide obtained from locust lipophorin (Nagao et al., 1987).
Galactose-containing oligosaccharides
N-Glycans G2 (100.4), I (110.4F), and K (110.4), which were distinct to T.ni cells overexpressing GalT, were all observed to contain terminal Gal residues. The identification of the structures of glycans G2 and K was not difficult due to the availability of the known references, however, there was no available reference for the structure of glycan I.
After sequential digestion by ß-galactosidase and ß-N-acetylhexosaminidase, the coordinate on the 2-D map of glycan I finally coincided with that of glycan H (Figure 6). Furthermore, the pathway profiles obtained by digestion with ß-galactosidase and ß-N-acetylhexosaminidase from glycan I to H, from glycan K to J2 via J3, and from glycan G2 to F2 via F3 were completely parallel (Figure 6). These results strongly suggest that the Gal-GlcNAc sequence in glycan I is linked to the Man3Manß4 antenna, because if the Gal-GlcNAc is linked to the Man6Manß4, the glycosidase digestion profile follows a different pathway (Tomiya and Takahashi, 1998). In addition, after prolonged -fucosidase digestion, glycan I was converted to glycan G2. Therefore, the proposed structure of glycan I is as follows:
|
The tandem MS spectrum of the parent ion (m/z 823.8) gives a fragmentation pattern which is consistent with the structure of glycan I (Figure 7). As shown in Figure 7, daughter ions were due to fragmentation at glycosidic bonds. In particular, the appearance of the ion peak at 1281.6 produced from the original molecule by loss of the Hex1HexNAc1 portion, suggests the existence of a Gal-GlcNAc sequence in the nonreducing end of the lower antenna. The peak ion 366.2 can be interpreted as the relative molecular mass of Gal-GlcNAc, which complements 1281.6. Subsequently, one Fuc, one mannose, and two Fuc residues are individually lost from the 1281.6 ion and result in the corresponding ions, 1135.5, 1119.5, and 989.4, respectively. By contrast, from the reducing end of glycan I, each ion 300.2, 446.3, and 592.3 is produced and corresponds to the presence of fragments GlcNAc-PA, fucosyl GlcNAc-PA, and difucosyl GlcNAc-PA, respectively. Other small daughter ions are consistent with the loss of each monosaccharide from the glycan I. Thus, ESI-tandem MS data confirmed that glycan I has a Gal-GlcNAc sequence at the nonreducing end and two Fuc residues at the reducing terminal GlcNAc.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
|---|
Western blot and Coomassie stain analyses demonstrated that recombinant hTf was expressed and secreted from insect cells. However, the MW of hTf produced in insect cells is lower than that of the native protein. Native hTf contains two N-glycosylation sites that are predominantly occupied by terminally sialylated biantennary complex oligosaccharides (Spik et al., 1975
). Metabolic radiolabeling of insect cells infected with the recombinant hTf baculovirus reveals the presence of 3 MW bands representing multiple hTf glycoforms in whole cell lysates. These glycoforms may represent site occupancy variants of hTf where zero, one, and two sites have been glycosylated. Treatment of the insect cells with tunicamycin collapsed these glycoforms to the lowest MW variant, presumably that of nonglycosylated hTf. Other heterologous glycoproteins have been previously found in glycosylated and nonglycosylated forms in insect cells following baculovirus infection (Kawamoto et al., 1994; Tate and Blakely, 1994; Hooker et al., 1999). The downward shift in MW of secreted hTf when treated with tunicamycin suggests that much of the secreted hTf contains oligosaccharides of some type. HTf carbohydrates have been previously shown to have no effect on iron binding, iron transport (Spik et al., 1988), and receptor binding (Mason et al., 1993), but they may be important during the folding and secretory processing.
Analysis of secreted hTf N-glycans by three-dimensional HPLC revealed the presence of high mannose, paucimannosidic, and some hybrid oligosaccharide structures. The composition (mol%) of high mannose, paucimannose, and hybrid structures from T.ni cells in the absence of GalT was 30.8%, 54.0%, and 13.9%, respectively, whereas the levels were 62.3%, 17.6%, and 16.4%, respectively, in the presence of GalT. High mannose structures are commonly found in heterologous glycoproteins from insect cells and may be the result of product secretion or cell lysis. At harvest time, the cell culture infected with both hTF and GalT baculoviruses had a lower viability than the culture infected with the hTF virus only, which was expected from the higher MOI used to infect cells with the two baculoviruses (Kioukia et al., 1995). An increase in lysis of the coinfected cells may result in an increase in the release of intracellular glycoproteins. We have previously observed that intracellular glycoforms include a higher percentage of high mannose structures (Hsu et al., 1997). A higher percentage of lysed cells would result in a release of these partially processed high mannose glycoforms into the medium.
An examination of the high mannose structures provides insights into the processing of oligosaccharides in insect cells. The proposed oligosaccharide processing pathway (Figure 8) appears to follow the same general pathway observed in mammalian cells (Kornfeld and Kornfeld, 1985; Marchal et al., 1999); however, it is possible that alternative pathways may exist in insect cells. The presence of the multiple Man8GlcNAc2, Man7GlcNAc2, and Man6GlcNAc2 isomers suggests that the order of mannose residue cleavage during mannosidase processing is variable. Alternatively, the accumulated high-Man types may have been subjected to degradative -mannosidase digestions. In addition, one structure, M9.2, is observed which includes an attached glucose. M9.2 is a structure found early in the oligosaccharide processing pathway and is the product of glucose cleavage reactions of the Glc3Man9GlcNAc2 core by glucosidases. Alternatively, M9.2 may be a product of re-glucosylation by a previously identified insect glucosyltransferase (Parker et al., 1995) in the cycle of reglucosylation that occurs prior to transport of folded proteins from the ER (Sousa and Parodi, 1995; Wada et al., 1997).
|
In addition to the high mannose structures were significant levels of paucimannosidic structures and some hybrid structures. Hybrid structures including GlcNAc on both the
(1,3)- and (1,6)-Man branches were observed in both cultures, confirming the presence of native GlcNAcTI and GlcNAcTII activities in T.ni cells. In the absence of GalT expression, however, much of the GlcNAc present on the (1,3)-Man branch appears to be removed by N-acetylglucosaminidase activity which has been found to be present in insect cells (Licari et al., 1993; Altmann et al., 1995). Also observed, in some cases, is the presence of Fuc on the reducing GlcNAc, which, when (1,3) linked, represents a potentially allergenic glycoform produced by insect cells (Aalberse et al., 1981; Tretter et al., 1993).
Examination of the oligosaccharide structures from hTf produced in cells infected with the GalT baculovirus revealed that transferase overexpression can indeed alter the glycoforms of the expressed transferrin. Three hybrid type oligosaccharides (100.4, 110.4F, 110.4) bearing terminal Gal moieties were identified in hTf from T.ni insect cells coinfected with the GalT baculovirus that were not detected in cells that were infected with AcNPV-hTf alone. Interestingly, over 80% of oligosaccharides containing GlcNAc on the (1,3)-Man arm also include a terminal Gal residue. Sf-9 cells infected with the GalT baculovirus (Wolff et al., unpublished observations) or stably transformed to express GalT (Hollister et al., unpublished observations) were also observed to produce hybrid N-glycans terminating in galactose. In one of these studies, a higher percentage of Gal terminating structures was obtained from a recombinant gp64 fusion protein (Hollister et al., unpublished observations). Differences in the levels of galactosylation may be attributable to a number of factors including the particular glycoprotein, the host cell line, cell lysis effects, and GalT enzyme activity during the infection period (Jarvis et al., 1996).
Of the Gal containing carbohydrate structures, it appears the GalT activity was exclusive to the (1,3)-Man branch. The absence of Gal residues on the (1,6)-Man branch may result from low levels of GlcNAc bearing acceptor substrate or the overexpressed mammalian galactosyltransferase may have a high specificity for the (1,3)-Man arm. When GlcNAc2Man3GlcNAc2 was used as an acceptor substrate, several mammalian galactosyltransferases where shown to demonstrate a preference for the (1,3)-Man arm (Blanken et al., 1984, Paquet et al., 1984; Narasimhan et al., 1985). Alternatively, it is possible that the recombinant GalT is expressed in a cellular compartment prior to the addition of GlcNAc by homologous GlcNAcTII. However, recent evidence suggests that recombinant GalT is localized in the correct organelle (i.e., trans Golgi) of Sf-9 cells (Kawar and Jarvis, unpublished observations).
In addition to enhancing galactosylation, GalT overexpression appeared to inhibit the formation of paucimannosidic products. The percentage of hybrid oligosaccharide structures as a total of hybrid and paucimannosidic structures increased from 20% without GalT to 48% in cells overexpressing GalT. The decrease in the amount of paucimannosidic structures and the concomitant increase in Gal containing hybrid structures in the hTf N-glycans from cells overexpressing GalT may be due to competition between GalT and ß-N-acetylglucosaminidase activities. The transfer of Gal to ß(1,2)-GlcNAc of the 1,3-Man arm may prevent cleavage of ß(1,2)-GlcNAc by the previously identified insect ß-N-acetylglucosaminidase.
However, even with GalT overexpression, no complex biantennary structures were observed attached to the recombinant transferrin. In a previous study, approximately 36 mol% of oligosaccharide structures obtained from the secreted fraction of a recombinant IgG produced in T.ni cells were of the complex type, some including Gal residues (Hsu et al., 1997). Final N-glycan structures may be influenced by the specific polypeptide chain undergoing N-glycosylation (Anderson and Goochee , 1994; Pfeiffer et al., 1994) as well as by differences in the cell culture conditions (Anderson and Goochee, 1994). The activity of glycosidases released into the culture medium following cell lysis (Parker et al., 1995) may also alter the N-glycan structures of extracellular glycoproteins.
Clearly, the overexpression of GalT enhances the level of oligosaccharide complexity for hTf expressed in T.ni cells. Overexpression of GalT may overcome a limitation in constitutive insect cell GalT activity and/or compete with N-acetylglucosaminidase activity to alter the N-linked oligosaccharide processing pathway. Furthermore, these studies also indicate that several other processing steps, including the addition of GlcNAc on the (1,6)-Man branch, could possibly be altered using oligosaccharide modifying enzymes to generate a product which is more similar in composition to native transferrin.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
|---|
Materials
Trichoplusia ni (T.ni, BTI-TN5B1-4, High FiveTM) and the TA cloning kit were obtained from Invitrogen (Carlsbad, CA). Sf-9 cells and the plasmid containing the gene for human transferrin (TFR27A) were obtained from the American Type Culture Collection (Manassas, VA). Excell 401 and 405 medium was purchased from JRH Biosciences (Lenexa, KS). Oligonucleotides used in the PCR reaction were obtained from Integrated DNA Technologies (Coralville, IA). Taq polymerase was purchased from Stratagene (La Jolla, CA). Restriction enzymes, DH5
competent cells, agarose, and 5-bromo- 4-chloryl-3-indoyl-ß-D-galactopyranoside (X-gal) were obtained from Life Technologies (Gaithersburg, MD). Baculovirus transfer plasmid and linear DNA were obtained from Clonetech (Palo Alto, CA). Qiagen (Valencia, CA) QIAEX kit was used to purify DNA from agarose gels. Hydrochloric acid, ammonium sulfate, and sodium citrate were purchased from J.T.Baker (Phillipsburg, NJ). Coomassie brilliant blue, Tween-20, nitrocellulose membrane, and Bio-Gel P-4 were obtained from Bio-Rad (Hercules, CA). Glycoamidase A (glycopeptidase A, EC 3.5.1.52) from sweet almond (Takahashi, 1977), -mannosidase, ß-galactosidase, and ß-N-acetylhexosaminidase from jack bean were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). -L-Fucosidase from bovine kidney was purchased from Boehringer Mannheim (Mannheim, Germany). The pyridylamino (PA) derivatives of isomalto-oligosaccharides (4–20 glucose residues) and of reference oligosaccharides (code nos. 000.1, 010.1, 100.2, 100.4, 110.1, 110.2, 110.4, M5.1, M6.1, M7.1, M7.2, M8.1, M8.2, and M.9.1) were from Seikagaku Kogyo. Code no. 010.0 was obtained by partial -mannosidase digestion of 010.1. Code no. M6.10 was similarly obtained by partial -mannosidase digestion of M9.1 (Tomiya et al., 1991). M9.2 was obtained from locust lipophorin (Nagao et al., 1987). Code no. 010.1F was obtained from neuropsin produced in T.ni cells (Takahashi et al., unpublished observations). The following materials were obtained from the sources indicated: Dialysis tubing and peroxidase labeled anti-IgG antibody, from Pierce (Rockford, IL); anti-human transferrin antibody, Dako (Carpinteria, CA); Lumiglo, Kirkegaard & Perry (Gaithersburg, MD); Tran35S-label, ICN (Costa Mesa, CA); Amplifier, Amersham Pharmacia Biotech (Piscataway, NJ); PVDF membrane, Millipore (Bedford, MA); Dowex 50W-X8, Dow Chemical Co. (Midland, MI); sodium cyanoborohydride, Aldrich (Milwaukee, WI), 2-aminopyridine, Wako Pure Chemical Industries (Osaka, Japan). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.
Cell culture and virus stock
Suspension cultures of T.ni and Sf-9 cells were maintained at 27°C and rotated at 120 r.p.m. in 250 ml shaker flasks containing 35 ml of Excell 405 and Excell 401 serum-free medium, respectively. For protein purification, 500 ml medium in 1 l shaker flasks were maintained as stated above.
The recombinant baculovirus (AcP(+)IE1GalT) encoding the gene for bovine GalT was constructed as previously described (Jarvis and Finn, 1996). The recombinant baculovirus encoding the gene for hTf was constructed as described below.
Baculovirus construction
Recombinant hTf was amplified from plasmid TFR27A by polymerase chain reaction using Taq polymerase. The 5' and 3' primers were GCG CCC TCG AGA TGA GGC TCG CCG T and CCT ACC TTC TAG ATT TTA AGG TCT ACG GAA, respectively. The 5' and 3' primers contain XhoI and BglII restriction sites, respectively, for subsequent cloning into the baculovirus transfer vector. The amplified gene was ligated into a linear cloning vector (PCRII) and transformed into E.coli using the TA cloning kit. Following screening for the hTf insert, hTf positive colonies were miniprepped and the plasmid DNA digested with XhoI and BglII restriction enzymes. The digested hTf gene fragment was purified from a 1% agarose TAE gel using a QIAEX DNA extraction kit. The purified and digested gene was ligated into pBacPAK8TM, a baculovirus transfer vector which was also digested with XhoI and BglII restriction enzymes and gel purified. The resultant ligation reaction mixture was transformed into E.coli DH5 competent cells. E.coli colonies grown on LB (w/100 µg/ml ampicillin) agar plates were cultured, miniprepped, and screened for plasmids containing the hTf insert.
Plasmid DNA containing hTf (pBachTf) was transfected with BacPAK6, a linear baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) genomic DNA fragment, into Sf-9 insect cells. Recombinant baculovirus was identified by selecting clear plaques in low melting point agarose containing X-gal. HTf positive plaques were amplified in Sf-9 suspension cultures to provide working stocks of the recombinant hTf baculovirus (AcNPV-hTf).
Cell infection and harvest
Log phase insect cells were seeded in a 6 well plate at 1 x 106 cells/well (1.5–2 ml). Cells were allowed to attach to the well (20 min at 27°C) and infected with AcNPV-hTf at a multiplicity of infection (MOI) of 5 to ensure synchronous infection. At 72 h postinfection (pi), infected cells were collected with medium in 1.5 ml microfuge tubes and centrifuged 30 s at 16,000 x g. After removal of the medium, the cell pellets were incubated with 300 µl lysis buffer (1% Triton X-100, 50 mM Tris-HCl pH 8.5, 10 mM EDTA, 150 mM NaCl) and protease inhibitors (0.5 µg/ml antipain, 0.5 µg/ml aprotinin, 15.6 µg/ml benzamidine-HCl, 0.5 µg/ml chymostatin,1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl). Clarified lysates were obtained from crude whole cell lysates by centrifugation for 15 min at 16,000 x g at 4°C. The medium from the infected cells was used to evaluate secreted hTf expression levels.
Large scale suspension cultures were infected with AcNPV-hTf alone or with AcNPV-hTf and AcP(+)IE1GalT at a density of 2 x 106 cells/ml. Two hours after infection fresh medium was added to the culture to adjust the cell density to ~1 x 106 cells/ml. At 66 h pi the cultures were centrifuged 20 min at 4000 x g to collect the medium which contains the secreted hTf.
Coomassie stain and Western blot
An equivalent number of cells from whole cell and clarified lysates from infected insect cells were separated by SDS–PAGE (10%) using reducing (60 mM Tris–HCl [pH 6.8], 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue) sample buffer. Following electrophoresis, the gel was stained with 0.1% Coomassie brilliant blue stain to visualize protein bands. Secreted hTf was similarly analyzed using an equivalent volume of medium.
For Western blotting, the gel was transferred onto a nitrocellulose membrane following electrophoresis. The membrane was blocked 2 h in a 10% (w/v) dried milk Tris-buffered saline with 0.05% Tween 20 detergent (TBST) solution. Recombinant hTf was detected by probing with a primary polyclonal rabbit anti-human hTf antibody for 1 h in TBST containing 2% dried milk. The primary antibody was followed by a 1 h incubation in TBST with a horse radish peroxidase (HRP) conjugated polyclonal goat anti-rabbit IgG. Protein bands were visualized using LumiGLOTM substrate.
35S-Methionine metabolic labeling
T.ni cells were seeded and infected in a 24-well plate at a density of 2 x 105 cells/well in the presence or absence of 1 µM tunicamycin. At 44 h postinfection the medium was aspirated and the cells were washed once with methionine-deficient Excell 401. The cells were then incubated with 50 µCi/ml of Tran35S-label methionine for 4 h. At the end of the labeling period, the cells were harvested and lysed, and whole cell lysates were subjected to SDS–PAGE as described above. The collected medium was incubated with 25 µl of protein-A Sepharose beads in the presence of 10 µl of polyclonal mouse anti-transferrin and 2 µl of polyclonal rabbit anti-mouse IgG antibodies overnight at 4°C with constant rotation. The beads were then washed twice with 700 µl of cold PBS and boiled 5 min in 40 µl of 2x sample buffer to elute the hTf prior to SDS–PAGE. The radioactive bands on the SDS–PAGE gel were visualized by autoradiography after soaking the gels in Amplifier fluorographic reagent for 20 min and vacuum drying. Kodak BMX x-ray film was used to record the images.
Recombinant hTf purification
Secreted hTf was purified from the insect cell medium using the strategy developed by Ali et al. (1996). Following collection, the medium was concentrated 10X using a hollow fiber 5000MWC ultrafiltration membrane (A/G Technology, Needham, MA). The concentrated medium was diluted 50% with a 2.4 M ammonium sulfate/0.8 M sodium citrate buffer (pH 6), centrifuged, and filtered to remove the particulate matter. The sample was loaded on a phenyl Sepharose 6 fast flow column and washed with 1.2 M ammonium sulfate/0.4 M sodium citrate buffer (pH 6)(buffer A), and the proteins were eluted with a 0–100% water gradient in buffer A using a Pharmacia FPLC system (Amersham Pharmacia Biotech. The fractions containing recombinant hTf in high purity (as determined by Coomassie stain) were pooled together, dialyzed against 20 l of water overnight in membrane tubing (7000 MWCO), and lyophilized.
Monosaccharide analysis
The lyophilized hTf samples were resuspended in 50 mM ammonium acetate or phosphate buffered saline. Approximately 400 µg (200 µg/gel) of each hTf sample from T.ni cell cultures was subjected to SDS–PAGE (7.5%) and transferred to a PVDF membrane. Following transfer, the gel was stained with 0.1% Coomassie brilliant blue and visually compared to a nontransferred control to determine the efficiency of hTf transfer to the PVDF membrane. The membrane was stained with 0.1% Coomassie brilliant blue to visualize the protein bands. The protein bands corresponding to hTf were cut from the membrane and placed in pretreated (4 M HCl overnight at 100°C) 1.5 ml vials (2 vials/sample) and rinsed with double distilled water. The membranes were submerged and hydrolyzed in 2 M trifluoroacetic acid (TFA) for 4 h or 4 M HCl for 6 h at 100°C. Following hydrolysis and removal of the membrane, 6 nm 6–0-methyl-D-galactose was added to the samples as a control and the samples were dried using a Speed Vac. The dried samples were dissolved in double distilled water, dried, and dissolved in 250 µl water. The TFA hydrolyates were then passed through a 2 ml bed volume of Dowex 50-X8 resin (prewashed with 2 M HCl and rinsed with water) to remove the charged molecules including amino sugars. The eluent (~6 ml) was dried and dissolved in 250 µl water. Each sample (50 µl) was then passed through a CarboPac PA-1 column (Dionex) and analyzed for monosaccharide composition using High Performance Anion Exchange Chromatography with pulsed amperometric detection (HPAEC/PAD) on a Dionex BioLC (Fan et al., 1994). Monosaccharides were eluted isocratically with 7% NaOH.
Preparation and derivatization of N-glycans from recombinant transferrin
Transferrin produced in insect cells without (9 mg of protein correspond to 240 nmol of oligosaccharides) and with (4 mg of protein correspond to 107 nmol of oligosaccharides) GalT expression was heated at 100°C for 10 min. After digestion of the transferrin glycoprotein with trypsin and chymotrypsin (each 1%, w/w, of the substrate protein), at pH 8.0, the peptide/glycopeptide mixture was treated with glycoamidase A (0.6 mU) in 30 µl of 0.5 M citrate/phosphate buffer at pH 4.0 for 16 h to release the oligosaccharides. The mixture was finally digested with 1% (w/w) of pronase to convert peptide materials to amino acids and small peptides. The supernatant was applied to a column of Bio-Gel P4 (1x40 cm) equilibrated in 10 mM ammonium carbonate. The effluent was monitored using A280nm fluorescence and 1 ml fractions were collected which were then thoroughly dried (Nakagawa et al., 1995). After reductive amination with 2-aminopyridine using sodium cyanoborohydride (Yamamoto et al., 1989), the resultant PA-oligosaccharides were purified by gel filtration on a Sephadex G-15 column (1.0 x 40 cm).
Isolation and characterization of PA-oligosaccharides by three successive HPLC steps
All separations were performed on a LC-10A HPLC system (Shimadzu, Japan). Approximately a 1/200 portion from each of the PA-oligosaccharide mixtures was separated and characterized by HPLC using three-dimensional sugar mapping technique as described previously (Takahashi et al., 1995). PA-oligosaccharides were successively separated on an anion exchange column, TSKgel DEAE-5PW (7.5 x 75 mm; TOSOH, Japan), a reversed-phase column (Shim-pack CLC-ODS 6 x 150 mm, Shimadzu, Japan), and an amide-adsorption column (Amide-80, 4.6 x 250 mm, TOSOH). Elution conditions of the three columns were as described previously (Takahashi et al., 1995; Tomiya et al., 1988). The elution time for each peak was recorded in glucose units (Glc Units) and plotted on the x axis (for the reversed-phase column) and y axis (for the amide-adsorption column). After plotting the x and y coordinates for all PA-oligosaccharides on a two-dimensional (2-D) map, we compared the coordinate of a given sample with those of known PA-oligosaccharides. Each of the sample PA-oligosaccharides was co-chromatographed with a reference PA-oligosaccharide on ODS and amide columns to confirm its identity. The sample and reference PA-oligosaccharides were also digested simultaneously with several exoglycosidases and their coordinates were again compared on the map. PA-oligosaccharides were detected by fluorescence using excitation and emission at 320 and 400 nm, respectively.
Exoglycosidase digestion procedure
Each PA-oligosaccharide (50 pmol) isolated from the successive chromatography was digested with exoglycosidases (ß-galactosidase, ß-N-acetylhexosaminidase, and -L-fucosidase) under conditions described previously (Takahashi and Tomiya, 1992). The elution coordinate of each exoglycosidase-trimmed oligosaccharide was examined on the 2-D map to verify its structural identity.
MALDI-MS analysis
The molecular mass of PA-oligosaccharides were determined by MALDI mass spectrometry using a Voyager Elite time-of-flight mass spectrometer equipped with a delayed-extraction system (PerSeptive Biosystems, Framingham, MA). The samples were ionized by a nitrogen laser at 337 nm. 2,5-Dihydroxybenzoic acid was dissolved in 50% aqueous acetonitrile solution containing 0.1% trifluoroacetic acid at a concentration of 10 mg/ml. The samples were dissolved in distilled water and mixed with the matrix solution at a 1:5 volume ratio. One microliter of aliquot was placed on the sample plate and air dried. The data was analyzed using GRAMS/386 software.
ESI-MS and tandem MS analysis
Electrospray MS and tandem MS data were acquired using a Q-TOF instrument (Micromass, Manchester, UK) and MassLynx data acquisition. This hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer was fitted with a nanoflow electrospray ion source. Samples were dissolved in 50% aqueous methanol solution containing 0.2% formic acid and loaded into a nanoflow tip. A high voltage (1–1.3 kV) was applied to the nanoflow tip of the capillary. The collision energy was increased to an optimum value of 15–30 V using argon collision gas.
|
Abbreviations |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
|---|
GlcNAcTI, ß(1,2)-N-acetylglucosaminyltransferase I; II GlcNAcTII, ß(1,2)-N-acetylglucosaminyltransferase; GlcNAc, N-acetylglucosamine; GalT, ß(1,4)-galactosyltransferase; Gal, galactose; Fuc, fucose; IgG, immunoglobulin G; T.ni, Trichoplusia ni insect cell line; Sf-9, Spodoptera frugiperda insect cell line; hTf, human serum transferrin; PA, pyridylamino; MOI, multiplicity of infection; pi, postinfection; MW, molecular weight; MS, mass spectrometry.
|
Footnotes |
|---|
1 To whom correspondence should be addressed
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
|---|
Aalberse,R.C, Koshte,V. and Clemens,J.G.J. (1981) Cross-reactions between vegetable foods, pollen and bee venom due to Ige antibodies to a ubiquitous carbohydrate determinant. Int. Archs. All. Appl. Immunol., 66, 259–260.
Aisen,P. (1989) Physical biochemistry of the transferrins: update, 1984–1988. In Loehr,T.M. (ed.), Iron Carriers and Iron Proteins. VCH Publishers, New York, pp. 353–371.
Ali,S.A., Joao,H.C., Csonga,R., Hammerschmid,F. and Steinkasserer,A. (1996) High-yield production of functionally active human serum transferrin using a baculovirus expression system and its structural characterization. Biochem. J., 319, 191–195.
Altmann,F. (1996) N-Glycosylation in insects revisited. Trends Glycosci. Glycotech., 8, 101–114.
Altmann,F., Kornfeld,G., Dalik,T., Staudacher,E. and Glossl,J. (1993) Processing of asparagine-linked oligosaccharides in insect cells. N-acetylglusoaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology, 3, 619–625.
Altmann,F., Schwihla,H., Staudacher,E., Glossl,J. and Marz,L. (1995) Insect cells contain an unusual, membrane-bound ß-N-acetylglucosaminidase propably involved in the processing of protein N-glycans. J. Biol. Chem., 270, 17344–17349.
Anderson,D.C. and Goochee,C.F. (1994) The effect of cell-culture conditions on oligosaccharide structures of secreted glycoproteins. Curr. Opin. Biotech., 5, 546–549.[Medline]
Bartfeld,N.S. and Law,J.H. (1990) Isolation and molecular cloning of transferrin from the tobacco hornworm, Manduca sexta. J. Biol. Chem., 265, 21684–21691.
Blanken,W.M., van Vliet,A. and van den Eijnden,D.H. (1984) Branch specificity of bovine colostrum and calf thymus UDP-Gal: N-acetylglucosaminide beta-1,4-galactosyltransferase. J. Biol. Chem., 259, 15131–15135.
Bullen,J.J., Ward,C.G. and Rogers,H.J. (1990) Iron, infection and the role of bicarbonate. FEMS Microbiol. Lett., 71, 27–30.
Butters,T.D. and Hughes,R.C. (1981) Isolation and characterization of mosquito cell membrane glycoproteins. Biochim. Biophys. ACTA, 640, 655–671.[Medline]
Chen,W. and Bahl,O.P. (1991) Recombinant carbohydrate and selenomethionyl variants of human choriogonadotropin. J. Biol. Chem., 266, 8192–8197.
Davidson,D.J., Fraser,M.J. and Castillino,F.J. (1990) Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells. Biochemistry, 29, 5584–5590.[Medline]
Fan,J.-Q., Namiki,Y., Matsuoka,K. and Lee,Y.C. (1994) Comparison of acid hydrolytic conditions for ASN-linked oligosaccharides. Anal. Biochem., 219, 375–378.[ISI][Medline]
Fu,D. and van Halbeek,H. (1992) N-Glycosylation site mapping of human serotransferrin by serial lectin affinity chromatography, fast atom bombardment-mass spectrometry and 1H nuclear magnetic resonance spectroscopy. Anal. Biochem., 206, 53–63.[ISI][Medline]
Hard,K., van Dorn,J.M., Thomas-Oates,J.E., Kamerling,J.P. and van der Horst,D.J. (1993) Structure of the ASN-linked oligosaccharides of apolipoprotein III from the insect Locusta migraforia. Carbohydrate-linked 2-aminoethylphosphonate as a constituent of a glycoprotein. Biochemistry, 32, 766–775.[Medline]
Hollister,J.R., Shaper,J.H. and Jarvis,D.L. (1998) Stable expression of mammalian ß1–4-galactosyltransferase extends the N-glycosylation pathway in insect cells. Glycobiology, 8, 473–480.
Hooker,A.D., Green,N.H., Baines,A.J., Bull,A.T., Jenkins,N., Strange,P.G. and James,D.C. (1999) Constraints on the transport and glycosylation of recombinant IFN-
in Chinese hamster ovary and insect cells. Biotech. Bioeng., 63, 559–572.
Hsieh,P. and Robbins,P.W. (1984) Regulation of asparagine-linked oligosaccharide processing. Oligosaccharide processing in Aedes albopitus mosquito cells. J. Biol. Chem., 259, 2975–2382.
Hsu,T.-A., Takahashi,N., Tsukamoto,Y., Kato,K., Shimada,I., Masuda,K., Whiteley,E., 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, 9062–9070.
Jarvis,D.L. (1997) Baculovirus Expression Vectors. In Miller,L.K. (ed.), The Baculoviruses. Plenum, New York, pp. 389–431.
Jarvis,D.L and Finn,E.E. (1996) Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nature Biotech., 14, 1288–1292.
Jarvis,D.L., Weinkauf,C. and Guarion,L.A. (1996) Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells. Pro. Expr. Purif., 8, 192–203.
Kawamoto,S., Hattori,S., Oiji,I., Hamajima,K., Mishina,M. and Okuda,K. (1994) Ligand-binding properties and N-glycosylation of alpha 1 subunit of the alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-selective glutamate receptor channel expressed in a baculovirus system. Eur. J. Biochem., 223, 665–673.[ISI][Medline]
Kioukia,N., Nienow,A.W., Emery,A.N. and al-Rubeai,M. (1995) Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus. J. Biotech., 38, 243–251.[ISI][Medline]
Klenk,H.-D. (1996) Post-translational modifications in insect cells. Cytotechnology, 20, 139–144.
Kornfeld,R. and Kornfeld,S. (1985) Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem., 54, 631–664.[ISI][Medline]
Kubelka,V., Altmann,F., Kornfeld,G. and Marz,L. (1994) Structures of the N-linked oligosaccharides of the membrane glycoproteins from three lepidopteran cell lines (Sf-21, IZD-Mb-0503, Bm-N). Arch. Biochem. Biophys., 308, 148–157.[ISI][Medline]
Kulakosky,P.C., Hughes,P.R. and Wood,H.A. (1998) N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells. Glycobiology, 8, 741–745.
Kuroda,K., Geyer,H., Geyer,R., Doerfler,W. and Klenk,H.D. (1990) The oligosaccharides of influenza virus hemmaglutinin expressed in insect cells by a baculovirus vector. Virology, 174, 418–329.[ISI][Medline]
Leger,D., Campion,B., Decottignies,J.-P., Montreuil,J. and Spik,G. (1989) Physiological significance of the marked increased branching of the glycans of human serotransferrin during pregnancy. Biochem. J., 257, 231–238.[ISI][Medline]
Licari,P.J., Jarvis,D.L. and Bailey,J.E. (1993) Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity. Biotech. Prog., 9, 146–152.[Medline]
Lindsay,D.A., Vonderfecht,S.L., Betenbaugh,M.J., Eiden,J.J. (1993) Baculovirus expression of gene 6 of the IDIR strain of group B rotavirus (GBR): coding assignment of the major inner capsid protein. Virology, 193, 367–375.[ISI][Medline]
Lopez,M., Coddeville,B., Langridge,J., Plancke,Y., Sautiere,P., Chaabihi,H., Chirat,F., Harduin-Lepers,A., Cerutti,M., Verbert,A. and Delannoy,P. (1997) Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells. Glycobiology, 7, 635–651.
Luckow,V.A. (1995) Protein production and processing from baculovirus expression vectors, In Shuler,M.L., Wood,H.A., Granados,R.R. and Hammer,D.A. (eds.), Bacuolvirus Expression Systems and Biopesticides. John Wiley & Sons, New York, pp. 51–90.
Luckow,V.A. and Summers,M.D. (1988) Trends in the development of baculovirus expression vector. Bio/Techniques, 6, 47–55.
MacGillivray,R.T.A., Mendes,E., Sinha,S.K., Sutton,M.R., Lineback-Zins,J. and Brew,K. (1983) The primary structure of human serum transferrin. J. Biol. Chem., 258, 3543–3546.
Marchal,I., Mir,A.-M., Kmiecik,D., Verbert,A. and Cacan,R. (1999) Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line. Glycobiology, 9, 645–654.
Marz,L., Altmann,F., Staudacher,E. and Kubelka,V. (1995) Protein glycosylation in insects. In Montreuil,J., Schachter,H. and Vliegenthart,J.F.G. (eds.), Glycoproteins. Elsevier Science, Amsterdam, pp. 543–563.
Mason,A.B., Miller,M.K., Funk,W.D., Banfield,D.K., Savage,K.J, Oliver,R.W.A., Green,B.N, MacGillivray,R.T.A. and Woodworth,R.C. (1993) Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins. Biochemistry, 32, 5472–5479.[Medline]
Montreuil,J., Spik,G. and Mazurier,J. (1997) Transferrin superfamily. In Montreuil,J., Vliegenthart,J.F.G. and Schacter H. (eds.), Glycoproteins II. Amsterdam: Elsevier. pp. 203–242.
Nagao,E., Takahashi,N. and Chino,H. (1987) Asparagine-linked oligosaccharides of locust lipohorin. Insect Biochem., 17, 531–538.
Nakagawa,H., Kawamura,Y., Kato,K., Shimada,I., Arata,Y. and Takahashi,N. (1995) Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. Anal. Biochem., 226, 130–138.[ISI][Medline]
Narasimhan,S., Freed,J.C. and Schachter,H. (1985) Control of glycoprotein synthesis. Bovine milk UDPgalactose:N-acetylglucosamine ß-4-galactosyltransferase catalyzes the preferential transfer of galactose to the GlcNAc ß 1,2Man 1,3- branch of both bisected and nonbisected complex biantennary asparagine-linked oligosaccharides. Biochemistry, 24, 1694–700.[Medline]
O’Reilly,D.R., Miller,L.K. and Luckow,V.A. (1992) Baculovirus expression vectors. W.H. Freeman & Company, New York.
Ogonah,O.W., Freedman,R.B., Jenkins,N., Kirit,P. and Rooney,B.C. (1996) Isolation and characterization of an insect cell line to perform complex N-linked glycosylation on recombinant proteins. Bio/Techniques, 14, 197–202.
Paquet,M.R., Narasimhan,S., Schachter,H. and Moscarello,M.A. (1984) Branch specificity of purified rat liver Golgi UDP-galactose: N-acetylglucosamine ß-1,4-galactosyltransferase. Preferential transfer of of galactose on the GlcNAc ß 1,2-Man 1,3-branch of a complex biantennary Asn-linked oligosaccharide. J. Biol. Chem., 259, 4716–4721.
Parker,C.G., Fessler,L.I., Nelson,R.E. and Fessler,J.H. (1995) Drosophila UDP-glucose:glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins. EMBO J., 14, 1294–1303.[ISI][Medline]
Pfeiffer,G., Strube,K.H., Schmidt,M. and Geyer,R. (1994) Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain. Eur. J. Biochem., 219, 331–348.[ISI][Medline]
Radmacher,T.W., Homans,S.W., Parekh,R.B. and Dwek,R.A. (1986) Immunoglobulin-G as a glycoprotein. Biochem. Soc. Symp., 51, 131–148.[Medline]
Salmon,V., Legrand,D., Georges,B., Slomianny,M.-C., Coddevelle,B. and Spik,G. (1997) Characterization of human lactoferrin produced in the baculovirus expression system. Prot. Expr. Purif., 9, 203–210.[ISI][Medline]
Sousa,M.C. and Parodi,A.J. (1992) The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. EMBO J., 14, 4196–4203.[ISI][Medline]
Spik,G., Bayard,B., Fournet,B., Strecker,G., Bouquelet,S. and Montreuil,J. (1975) Studies on glycoconjugates.64. complete structure of 2 carbohydrate units of human serotransferrin. FEBS Lett., 50, 296–299.[ISI][Medline]
Spik,G., Coddeville,B. and Montreuil,J. (1988) Comparative study of the primary structures of sero-, lacto- and ovotransferrin glycans from different species. Biochimie, 70, 1459–1469.[Medline]
Takahashi,N. (1977) Demonstration of a new amidase acting on glycopeptides. Biochem. Biophys. Res. Commun., 76, 1194–1201.[ISI][Medline]
Takahashi,N. and Tomiya,N. (1992) In Takahashi,N. and Muramatsu,T. (eds.), Handbook of Endoglycosidases and Glycoamidases. CRC Press, Boca Raton, pp. 199–332.
Takahashi,N., Nakagawa,H., Fujikawa,K., Kawamura,Y. and Tomiya,N. (1995) Three-dimensional elution mapping of pyridylaminated N-linked neutral and sialyl oligosaccharides. Anal. Biochem., 226, 139–146.[ISI][Medline]
Tandai,M., Endo,T., Sasaki,S., Masuho,Y., Kochibe,N. and Kubata,A. (1991) Structural study of the sugar moieties of monoclonal antibodies secreted by human-mouse hybridoma. Arch. Biochem. Biophys., 291, 339–348.[ISI][Medline]
Tate,C.G. and Blakely,R.D. (1994) The effect of N-linked glycosylation on activity of the Na (+)- and Cl (-)-dependent serotonin transporter expressed using recombinant baculovirus in insect cells. J. Biol. C