A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN^Lec1. We found that Sf-RVN and Sf-RVN^Lec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN^Lec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN^Lec1 cells were Endo H-cleavable Man₅GlcNAc₂ structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.     

Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III

The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3 ^+/+ and Mgat3 ^–/– mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3 ^–/– mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3 ^–/– mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3 ^–/– mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc. Published in 2003.     

Structural analysis of the glycosylation of gene-activated erythropoietin (epoetin delta, Dynepo)

Recently, a novel recombinant human erythropoietin (epoetin delta, Dynepo) has been marketed in the European Union for the treatment of chronic kidney disease, cancer patients receiving chemotherapy, and so forth. Epoetin delta is engineered in cultures of the human fibrosarcoma cell line HT-1080 by homologous recombination and “gene activation.” Unlike recombinant erythropoietins produced in other mammalian cells, epoetin delta is supposed to have a human-type glycosylation profile. However, the isoelectric focusing profile of epoetin delta differs from that of endogenous erythropoietin (both urinary and plasmatic). In this work, structural and quantitative analysis of the O- and N-glycans of epoetin delta was performed and compared with glycosylation from recombinant erythropoietin produced in Chinese hamster ovary (CHO) cells. From the comparison, significant differences in the sialylation of O-glycans were found. Furthermore, the N-glycan analysis indicated a lower heterogeneity from epoetin delta when compared with its CHO homologue, being predominantly tetraantennary without N-acetyllactosamine repeats in the former. The sialic acid characterization revealed the absence of N-glycolylneuraminic acid. The overall sugar profiles of both glycoproteins appeared to be significantly different and could be useful for maintaining pharmaceutical quality control, detecting the misuse of erythropoietin in sports, and establishing new avenues to link glycosylation with biological activity of glycoproteins.     

Reduction in Golgi apparatus dimension in the absence of a residential protein,N-acetylglucosaminyltransferase V

Various proteins are involved in the generation and maintenance of the membrane complex known as the Golgi apparatus. We have used mutant Chinese hamster ovary (CHO) cell lines Lec4 and Lec4A lacking N-acetylglucosaminyltransferase V (GlcNAcT-V, MGAT5) activity and protein in the Golgi apparatus to study the effects of the absence of a single glycosyltransferase on the Golgi apparatus dimension. Quantification of immunofluorescence in serial confocal sections for Golgi α-mannosidase II and electron microscopic morphometry revealed a reduction in Golgi volume density up to 49 % in CHO Lec4 and CHO Lec4A cells compared to parental CHO cells. This reduction in Golgi volume density could be reversed by stable transfection of Lec4 cells with a cDNA encoding Mgat5. Inhibition of the synthesis of β1,6-branched N-glycans by swainsonine had no effect on Golgi volume density. In addition, no effect on Golgi volume density was observed in CHO Lec1 cells that contain enzymatically active GlcNAcT-V, but cannot synthesize β1,6-branched glycans due to an inactive GlcNAcT-I in their Golgi apparatus. These results indicate that it may be the absence of the GlcNAcT-V protein that is the determining factor in reducing Golgi volume density. No dimensional differences existed in cross-sectioned cisternal stacks between Lec4 and control CHO cells, but significantly reduced Golgi stack hits were observed in cross-sectioned Lec4 cells. Therefore, the Golgi apparatus dimensional change in Lec4 and Lec4A cells may be due to a compaction of the organelle.     

Antibodies That Detect O-Linked β-d-N-Acetylglucosamine on the Extracellular Domain of Cell Surface Glycoproteins

The transfer of N-acetylglucosamine (GlcNAc) to Ser or Thr in cytoplasmic and nuclear proteins is a well known post-translational modification that is catalyzed by the O-GlcNAc transferase OGT. A more recently identified O-GlcNAc transferase, EOGT, functions in the secretory pathway and transfers O-GlcNAc to proteins with epidermal growth factor-like (EGF) repeats. A number of antibodies that detect O-GlcNAc in cytosolic and nuclear extracts have been described previously. Here we compare seven of these antibodies (CTD110.6, 10D8, RL2, HGAC85, 18B10.C7(#3), 9D1.E4(#10), and 1F5.D6 (#14) for detection of the O-GlcNAc modification on extracellular domains of membrane or secreted glycoproteins that may also carry various N- and O-glycans. We found that CTD110.6 binds not only to O-GlcNAc on proteins but also to terminal β-GlcNAc on the complex N-glycans of Lec8 Chinese hamster ovary (CHO) cells that lack UDP-Gal transporter activity and express GlcNAc-terminating, complex N-glycans. We show that CTD110.6, #3, and #10 antibodies can be used to detect cell surface glycoproteins bearing O-GlcNAc. Cell surface glycoproteins recognized by CTD110.6 antibody included NOTCH1 that possesses many EGF repeats with a consensus site for EOGT. Knockdown of CHO Eogt reduced binding of CTD110.6 to Lec1 CHO cells, and expression of a human EOGT cDNA increased the O-GlcNAc signal on Lec1 cells and the extracellular domain of NOTCH1. Thus, with careful controls, antibodies CTD110.6 (IgM), #3 (IgG), and #10 (IgG) can be used to detect membrane and secreted proteins modified by O-GlcNAc on EGF repeats.     

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.     

A Human Embryonic Kidney 293T Cell Line Mutated at the Golgi ��-Mannosidase II Locus

Disruption of Golgi α-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi α-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi α-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.Mammalian N-linked glycosylation is characterized by significant chemical heterogeneity generated by an array of competing glycosidases and glycosyltransferases (1). The structural analysis of recombinant glycoproteins, such as human erythropoietin (2, 3), has illustrated the capacity of mammalian expression systems for generating diverse N-linked glycans.Heterogeneity develops during egress of a glycoprotein through the secretory system (1). N-linked glycosylation is initiated in the rough endoplasmic reticulum (ER)⁴ by the co-translational transfer of Glc₃Man₉GlcNAc₂ to the asparagine residues of the glycosylation sequon. In the absence of protein misfolding, hydrolysis by ER α-mannosidase I plus α-glucosidase I and II results in the transfer of glycoproteins dominated by the D1,D3 isomer of Man₈GlcNAc₂ glycans to the Golgi apparatus (4). Further processing by Golgi α-mannosidases IA–C generates Man₅GlcNAc₂ (5–7), the principle substrate for UDP-N-acetyl-d-glucosamine:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (GnT I). The action of this enzyme yields classic hybrid-type glycans with mannosyl 6-antennae and processed 3-antennae (1). In the absence of the GnT III-mediated addition of bisecting GlcNAc, the two terminal α-mannose residues of the 6-antenna of hybrid-type glycans are cleaved by Golgi α-mannosidase II, forming mono-antennary complex-type glycans. These may then be processed by N-acetylglucosaminyltransferases, generating multiantennary complex-type glycans of enormous potential heterogeneity following the sequential transfer of monosaccharides such as galactose, N-acetylgalactosamine, fucose, and N-acetylneuraminic acid (8).The importance of this carbohydrate diversity in metazoan biology is illustrated by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. In humans, about 12 congenital disorders of glycosylation (CDG) have been identified with defects in the biosynthesis of N-linked glycans (9). One disorder characterized by changes in glycosylation is congenital dyserythropoietic anemia type II (hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS)) (10, 11). HEMPAS is a heterogenous autosomal recessive disorder that renders erythrocytes prone to lysis. Although the precise molecular basis of HEMPAS remains to be determined, it is characterized by either a reduction in β1→4-galactosyltransferase, GnT II, or, in some patients, Golgi α-mannosidase II activity (11, 12). Interestingly, the increase in cell surface terminal mannose in mice deficient in Golgi α-mannosidase II leads to autoimmunity through chronic activation of the innate immune system (13, 14).Lectin-resistant (Lec) cell lines harboring loss- or gain-of-function mutations affecting the biosynthesis of N-glycans have emerged as powerful tools for the investigation of these disorders (15). For example, genetic complementation using Lec2, containing a mutation in the cytosine monophosphate sialic acid transporter, was used to identify a novel CDG, type IIf (16). Lectin-resistant cell lines can also be used as hosts to study naturally occurring mutations, as in the case of CHO Lec23 cells used to screen α-glucosidase I mutations in CDG, type IIb (17). Other applications of lectin-resistant cell lines include the expression of specific glycoforms of therapeutic glycoproteins. Manipulating the structure of their carbohydrate moieties modulates the pharmacological properties of glycoproteins by altering their bioactivity, serum half-life, and/or tissue tropism (18). For example, β-glucocerebrosidase expressed in CHO Lec1 cells (deficient in GnT I activity) exhibits mannosylation and improved macrophage uptake for the treatment of Gaucher disease (19). Lectin-resistant CHO cell lines have also been used to improve the crystallizability of glycoproteins for structural determination by x-ray crystallography (20–24).The expression of therapeutic glycoproteins as one or more defined “glycoforms” is essential for their optimization and may even be necessary to obtain regulatory approval (25). To this end, eukaryotic expression systems have been developed that allow glycosylation to be controlled. Recently, Pichia pastoris-based strains with human glycosyltransferases have been established, allowing the expression of glycoforms with oligomannose-, hybrid-, and some complex-type glycans (26, 27) and even sialylated complex-type structures (28). However, mammalian expression remains the dominant technology in industrial settings, presumably because of its reliability for the expression of human secreted glycoproteins.Although the majority of lectin-resistant cell lines have been generated using CHO cells, no Golgi α-mannosidase II-deficient CHO cell line has been generated thus far (15). Furthermore, only one human lectin-resistant cell line, i.e. GnT I-deficient (Lec1) HEK 293S cells (29), has been produced. Hybrid-type glycosylation has been reported to accumulate in ricin-resistant baby hamster kidney cells (30, 31); however, these cells contain a reduced but detectable level of cell-surface complex-type glycans, consistent with an incomplete ablation of Golgi α-mannosidase II activity (31). Moreover, the hybrids from one of these lines contain a trimannosyl rather than pentamannosyl core and appear to be heavily influenced by GnT II deficiency, resulting in the formation of what are now commonly called monoantennary complex-type glycans (32–34). We now describe the isolation of an HEK 293T cell line mutated at the MAN2A1 locus and deficient in Golgi α-mannosidase II activity via selection with ricin.     

Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.     

Biochemical characterization of theO-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells

Alterations inN- andO-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. TheO-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoclonal antibodies to probe Western blots. TheO-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gal1-3GalNAc1-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human crythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAc1-Ser/Thr). Recombinant glycophorin A had the sameO-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect itsO-glycan composition. Deletion of theN-linked glycosylation site at Asn₂₆, by introducing the Mi.I mutation (Thr₂₈ Met) by site-directed mutagenesis, did not markedly affect theO-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells. This demonstrates that the presence or absence of theN-glycan did not influenceO-linked glycosylation of the recombinant glycoprotein. Finally, theO-glycans on recombinant glycophorin A expressed in the Lec 2 and Lec 8 glycosylation deficient CHO cells were characterized. TheO-glycans on Lec 2 cell glycophorin A were predominantly Gal1-3GalNAc1-Ser/Thr (T antigen), while those on Lec 8 glycophorin A were exclusively GalNAc1-Ser/Thr (Tn antigen). These results will lead to a better understanding of the cell biology and immunology of this important human erythrocyte glycoprotein.     

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.     

Carbohydrate heterogeneity of vesicular stomatitis virus G glycoprotein allows localization of the defect in a glycosylation mutant of CHO cells

The carbohydrate portion of the G glycoprotein of vesicular stomatitis virus (VSV) grown in CHO cells (CHO/VSV) has been fractionated on BioGelP6, concanavalin A-Sepharose, and pea lectin-agarose. The results suggest that, in addition to sialic acid and fucose heterogeneity, the asparagine-linked complex carbohydrate moieties of CHO/VSV also display branching heterogeneity. Although the majority of the glycopeptides bind to concanavalin A-Sepharose in a manner typical of certain biantennary carbohydrate structures, a significant proportion do not bind to the lectin. The latter behavior is typical of tri- or tetraantennary (branched) carbohydrate structures. The CHO/VSV glycopeptides which do not bind to concanavalin A-Sepharose separate into bound and unbound fractions on pea lectin-agarose suggesting that they include at least two different types of (branched) carbohydrate structures. Glycopeptides from the G glycoprotein of VSV grown in two, independently derived CHO glycosylation mutants which belong to complementation group 4 (Lec4 mutants) were examined in the same manner. In contrast to glycopeptides from CHO/VSV, glycopeptides from Lec4/VSV which passed through concanavalin A-Sepharose did not contain a component which subsequently bound to pea lectin-agarose. A glycopeptide fraction with these lectin-binding properties was also missing from cell surface glycopeptides derived from Lec4 cells. The combined results are consistent with the hypothesis that Lec4 CHO glycosylation mutants lack a glycosyltransferase activity responsible for the addition of a (branch) N-acetylglucosamine residue linked β1,6 to mannose.     

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.     

Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin,Especially Decreased Sialylation

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn¹⁶⁷. Highly sialylated O-glycans were found to be present in the Thr¹¹⁰–Thr¹²⁴ region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.     

Improved Method for Drawing of a Glycan Map,and the First Page of Glycan Atlas,Which Is a Compilation of Glycan Maps for a Whole Organism

Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.     

A subclass of cell surface carbohydrates revealed by a CHO mutant with two glycosylation mutations

A novel lectin-resistance phenotype was displayed by a LEC10 Chinese hamster ovary (CHO) cell mutant that was selected for resistance to the erythroagglutinin, E-PHA. Biochemical and genetic analyses revealed that the phenotype results from the expression of two glycosylation mutations, LEC10 and lec8. The LEC10 mutation causes the appearance of N-acetylglucosaminyltransferase III (GlcNAc-TIII) activity and the production of N-linked carbohydrates with a bisecting GlcNAc residue. The lec8 mutation inhibits translocation of UDP-Gal into the Golgi lumen and thereby dramatically reduces galactosylation of all glycoconjugates. This reduction in galactose addition does not, however, cause Lec8 mutants to be very resistant to the galactose-binding lectin, ricin. By contrast, the double mutant LEC10.Lec8 behaved like a LEC10 mutant and was highly resistant to ricin. Based on structural studies of cellular glycopeptides as well as glycopeptides of the G glycoprotein of vesicular stomatitis virus grown in mutant cells, it appears that the ricin resistance of LEC10.Lec8 cells is due to the presence of a small number of Gal residues on branched, N-linked carbohydrates that also carry the bisecting GlcNAc residue. Labelling of N-linked cellular carbohydrates with [3H]galactose was found to occur at a low level for a wide spectrum of cellular glycoproteins in independent Lec8 mutants. Studies of the LEC10.Lec8 mutant have, therefore, led to the identification of a subset of structures that are acceptors for Gal when intra-Golgi UDP-Gal levels are limiting. This mutant also illustrates the potential for regulating cell surface recognition by carbohydrate-binding proteins by altering the expression of a single glycosyltransferase such as GlcNAc-TIII.     

1H NMR spectroscopy of carbohydrates from the G glycoprotein of vesicular stomatitis virus grown in parental and Lec4 Chinese hamster ovary cells

Carbohydrate moieties derived from the G glycoprotein of Vesicular Stomatitis Virus (VSV) grown in parental Chinese hamster ovary (CHO) cells and the glycosylation mutant Lec4 have been analyzed by high-field ¹H NMR spectroscopy. The major glycopeptides of $\text{CHO}\text{VSV}$ and $\text{Lec4}\text{VSV}$ were purified by their ability to bind to concanavalin A-Sepharose. The carbohydrates in this fraction are of the biantennary, complex type with heterogeneity in the presence of α(2,3)-linked sialic acid and α(1,6)-linked fucose residues. A minor $\text{CHO}\text{VSV}$ glycopeptide fraction, which does not bind to concanavalin A-Sepharose but which binds to pea lectin-agarose, was also investigated by ¹H NMR spectroscopy. These carbohydrates are complex moieties which appear to contain N-acetylglucosamine in β(1,6) linkage. Their spectral properties are most similar to those of a triantennary complex oligosaccharide containing a 2,6-disubstituted mannose α(1,6) residue. Carbohydrates of this type are not found among the glycopeptides of VSV grown in the Lec4 CHO glycosylation mutant.     

Unusual <Emphasis Type="Italic">N</Emphasis>-type glycosylation of salivary prolactin-inducible protein (PIP): multiple Lewis<Superscript>Y</Superscript> epitopes generate highly-fucosylated glycan structures

Prolactin-inducible protein (PIP) is a glycoprotein found in body secretions from exocrine glands like saliva and seminal plasma. Important biological functions of PIP concentrations have been demonstrated, e.g. in tumor diagnosis and progression. PIP quantity has been also found useful to determine the success of chemotherapy of mammary carcinoma. Here, we present the analysis of the N-glycosylation of PIP isolated from different sources by LC-MS(/MS) and ¹H-NMR. We found a very uncommon N-type glycosylation of PIP in healthy individuals from both, seminal fluid and saliva. PIP carries unusual highly fucosylated N-linked glycans with multiple Lewis^y (Le^y) epitopes on bi-, tri- and tetraantennary structures resulting in up to nine fucosyl residues on a tetraantennary glycan. In most organs, Le^y epitopes are not present on N-glycans except in case of a tumor when it is highly up-regulated and important for prognosis. Here, for the first time on a specific glycoprotein Le^y antigens are unambiguously characterized on an N-type glycan by NMR spectroscopy. So far, for specific glycoproteins Le^y epitopes had only been reported on O-glycans. Furthermore, a correlation between a nonsynonymous single nucleotide polymorphism (SNP) and glycosylation pattern was detected: individuals heterozygous for the SNP causing the amino acid exchange ⁵¹Gln to ⁵¹His have glycan structures with a higher degree of sialylation compared to individuals lacking the SNP.     

Insulin receptors on Chinese hamster ovary (CHO) cells: altered insulin binding to glycosylation mutants

We have identified and characterized insulin receptors on Chinese hamster ovary (CHO) cells. Insulin binds in a time, temperature and pH dependent fashion and insulin analogues compete for 125I-insulin binding in order of their biological potencies. Furthermore, two CHO cell glycosylation mutants, B4-2-1, lacking high mannose containing glycoproteins, and Lec 1.3c, lacking complex carbohydrate containing glycoproteins, bind insulin with a much higher and lower affinity respectively than wild type cells. This is the first report of insulin receptors on CHO cells and the first to use glycosylation mutants to study the effects of abnormal carbohydrates on insulin binding.