Functional characterization of ECP-heparin interaction: a novel molecular model.

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.

The N-glycoform of sRAGE is the key determinant for its therapeutic efficacy to attenuate injury-elicited arterial inflammation and neointimal growth.

UNLABELLED: Signaling of the receptor for advanced glycation end products (RAGE) has been implicated in the development of injury-elicited vascular complications. Soluble RAGE (sRAGE) acts as a decoy of RAGE and has been used to treat pathological vascular conditions in animal models. However, previous studies used a high dose of sRAGE produced in insect Sf9 cells (sRAGE(Sf9))and multiple injections to achieve the therapeutic outcome. Here, we explore whether modulation of sRAGE N-glycoform impacts its bioactivity and augments its therapeutic efficacy. We first profiled carbohydrate components of sRAGE produced in Chinese hamster Ovary cells (sRAGE(CHO)) to show that a majority of its N-glycans belong to sialylated complex types that are not shared by sRAGE(Sf9). In cell-based NF-κB activation and vascular smooth muscle cell (VSMC) migration assays, sRAGE(CHO) exhibited a significantly higher bioactivity relative to sRAGE(Sf9) to inhibit RAGE alarmin ligand-induced NF-κB activation and VSMC migration. We next studied whether this N-glycoform-associated bioactivity of sRAGE(CHO) is translated to higher in vivo therapeutic efficacy in a rat carotid artery balloon injury model. Consistent with the observed higher bioactivity in cell assays, sRAGE(CHO) significantly reduced injury-induced neointimal growth and the expression of inflammatory markers in injured vasculature. Specifically, a single dose of 3 ng/g of sRAGE(CHO) reduced neointimal hyperplasia by over 70%, whereas the same dose of sRAGE(Sf9) showed no effect. The administered sRAGE(CHO) is rapidly and specifically recruited to the injured arterial locus, suggesting that early intervention of arterial injury with sRAGE(CHO) may offset an inflammatory circuit and reduce the ensuing tissue remodeling. Our findings showed that the N-glycoform of sRAGE is the key determinant underlying its bioactivity and thus is an important glycobioengineering target to develop a highly potent therapeutic sRAGE for future clinical applications. KEY MESSAGE: The specific N-glycoform modification is the key underlying sRAGE bioactivity Markedly reduced sRAGE dose to attenuate neointimal hyperplasia and inflammation Provide a molecular target for glycobioengineering of sRAGE as a therapeutic protein Blocking RAGE alarmin ligands during acute injury phase offsets neointimal growth.

Basic amino acid residues of human eosinophil derived neurotoxin essential for glycosaminoglycan binding.

Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore, in silico computer modeling and in vitro binding assays suggest critical roles for the following basic amino acids located within heparin binding regions (HBRs) of EDN 34QRRCKN39 (HBR1), 65NKTRKN70 (HBR2), and 113NRDQRRD119 (HBR3) and in particular Arg35, Arg36, and Arg38 within HBR1, and Arg114 and Arg117 within HBR3. Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN.

Liver-targeting of primaquine-(poly-γ-glutamic acid) and its degradation in rat hepatocytes.

We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20-80min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA-primaquine conjugate (TriGalNAc-PGA-PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250ng per million cells of the conjugate bound to one million rat hepatocytes at 0°C, and approximately 2µg per million cells of the conjugate was taken up over 7h incubation at 37°C. Furthermore, our results suggested that TriGalNAc-PGA-PQ was almost completely degraded over 24h, and small degradation products were secreted into cell culture medium. The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.

Elevated polyamines in urothelial cells from OAB subjects mediate oxotremorine-evoked rapid intracellular calcium rise and delayed acetylcholine release.

Increased polyamine signaling in bladder urothelial cells (BUC) may play a role in the pathophysiology of overactive bladder (OAB). We quantitated intracellular polyamine levels in cultured BUC from OAB and asymptomatic (NB) subjects. We assessed whether polyamines modulated rapid intracellular calcium ([Ca(2+)]i) changes and delayed acetylcholine (ACh) release evoked by oxotremorine (OXO, a muscarinic agonist). BUC were cultured from cystoscopic biopsies. High-performance liquid chromatography (HPLC) quantitated intracellular putrescine, spermidine, and spermine levels. Five-millimeter difluoromethylornithine (DFMO), and one-millimeter methylglyoxalbisguanylhydrazone (MGBG) treatments were used to deplete intracellular polyamines. Ten micrometers of OXO were used to increase [Ca(2+)]i levels (measured by fura 2 microfluorimetry) and trigger extracellular ACh release (measured by ELISA). Polyamine levels were elevated in OAB compared with NB BUC (0.5 ± 0.15 vs. 0.16 ± 0.03 nmol/mg for putrescine, 2.4 ± 0.21 vs. 1.01 ± 0.13 nmol/mg for spermidine, and 1.90 ± 0.27 vs. 0.86 ± 0.26 nmol/mg for spermine; P < 0.05 for all comparisons). OXO evoked greater [Ca(2+)]i rise in OAB (205.10 ± 18.82% increase over baseline) compared with in NB BUC (119.54 ± 13.01%; P < 0.05). After polyamine depletion, OXO evoked [Ca(2+)]i rise decreased in OAB and NB BUC to 43.40 ± 6.45 and 38.82 ± 3.5%, respectively. OXO tended to increase ACh release by OAB vs. NB BUC (9.02 ± 0.1 vs. 7.04 ± 0.09 µM, respectively; P < 0.05). Polyamine depletion reduced ACh release by both OAB and NB BUC. In conclusion, polyamine levels were elevated twofold in OAB BUC. OXO evoked greater increase in [Ca(2+)]i and ACh release in OAB BUC, although these two events may be unrelated. Depletion of polyamines caused OAB BUC to behave similarly to NB BUC.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells.

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man(5)GlcNAc(2)-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man(9)GlcNAc(2)-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man(5)GlcNAc(2)-PP-Dol through Glc(1)Man(9)GlcNAc(2)-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc(3)Man(9)GlcNAc(2)-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

An HPLC-MALDI MS method for N-glycan analyses using smaller size samples: application to monitor glycan modulation by medium conditions.

Existing HPLC methods can provide detailed structure and isomeric information, but are often slow and require large initial sample sizes. In this study, a previously established two-dimensional HPLC technique was adapted to a two-step identification method for smaller sample sizes. After cleavage from proteins, purification, and fluorescent labeling, glycans were analyzed on a 2-mm reverse phase HPLC column on a conventional HPLC and spotted onto a MALDI-TOF MS plate using an automated plate spotter to determine molecular weights. A direct correlation was found for 25 neutral oligosaccharides between the 2-mm Shim-Pack VP-ODS HPLC column (Shimadzu) and the 6-mm CLC-ODS column (Shimadzu) of the standard two- and three-dimensional methods. The increased throughput adaptations allowed a 100-fold reduction in required amounts of starting protein. The entire process can be carried out in 2-3 days for a large number of samples as compared to 1-2 weeks per sample for previous two-dimensional HPLC methods. The modified method was verified by identifying N-glycan structures, including specifying two different galactosylated positional isomers, of an IgG antibody from human sera samples. Analysis of tissue plasminogen activator (t-PA) from CHO cell cultures under varying culture conditions illustrated how the method can identify changes in oligosaccharide structure in the presence of different media environments. Raising glutamine concentrations or adding ammonia directly to the culture led to decreased galactosylation, while substituting GlutaMAX-I, a dipeptide of L-alanine and L-glutamine, resulted in structures with more galactosylation. This modified system will enable glycoprofiling of smaller glycoprotein samples in a shorter time period and allow a more rapid evaluation of the effects of culture conditions on expressed protein glycosylation.

Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core.

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

Expression of a functional Drosophila melanogaster CMP-sialic acid synthetase. Differential localization of the Drosophila and human enzymes.

CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12-24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes.

Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells.

Schneider 2 (S2) cells from Drosophila melanogaster have been used as a plasmid-based, non-lytic expression system for foreign proteins. Here, a plasmid encoding the human erythropoietin (hEPO) gene fused with a hexahistidine (His(6)) tag under the control of the Drosophila metallothionein (MT) promoter was stably transfected into Drosophila S2 cells. After copper sulfate induction, transfected S2 cells were found to secrete hEPO with a maximum expression level of 18 mg/L and a secretion efficiency near 98%. The secreted hEPO from Drosophila S2 had an apparent molecular weight of about 23-27 kDa which was significantly lower than a recombinant hEPO expressed in Chinese hamster ovary (CHO) cells (about 36 kDa). N-glycosidase F digestion almost completely eliminated the difference and resulted in the same molecular weight ( approximately 20 kDa) of de-N-glycosylated hEPO proteins. These data suggest that recombinant hEPO from S2 cells was modified with smaller N-glycans. Subsequently, the major N-glycans were identified following glycoamidase A digestion, labeling with 2-aminopyridine (PA), and two-dimensional high-performance liquid chromatography (HPLC) analysis in concert with exoglycosidase digestion. This analysis of N-glycans revealed that hEPO was modified to include paucimannosidic glycans containing two or three mannose residues with or without core fucose. A similar glycosylation pattern was observed on a recombinant human transferrin expressed in S2 cells. These results provide a detailed analysis of multiple N-glycan structures produced in a Drosophila cell line that will be useful in the subsequent application of these cells for the generation of heterologous glycoproteins.

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines.

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans.

Biosynthesis of human-type N-glycans in heterologous systems.

Insects, yeasts and plants generate widely different N-glycans, the structures of which differ significantly from those produced by mammals. The processing of the initial Glc2Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 in the endoplasmic reticulum shows significant similarities among these species and with mammals, whereas very different processing events occur in the Golgi compartments. For example, yeasts can add 50 or even more Man residues to Man(8-9)GlcNAc2, whereas insect cells typically remove most or all Man residues to generate paucimannosidic Man(3-1)GlcNAc2N-glycans. Plant cells also remove Man residues to yield Man(4-5)GlcNAc2, with occasional complex GlcNAc or Gal modifications, but often add potentially allergenic beta(1,2)-linked Xyl and, together with insect cells, core alpha(1,3)-linked Fuc residues. However, genomic efforts, such as expression of exogenous glycosyltransferases, have revealed more complex processing capabilities in these hosts that are not usually observed in native cell lines. In addition, metabolic engineering efforts undertaken to modify insect, yeast and plant N-glycan processing pathways have yielded sialylated complex-type N-glycans in insect cells, and galactosylated N-glycans in yeasts and plants, indicating that cell lines can be engineered to produce mammalian-like glycoproteins of potential therapeutic value.

Humanization of lepidopteran insect-cell-produced glycoproteins.

The insect cell-baculovirus expression vector system, widely used for glycoprotein production, is not ideal for pharmaceutical glycoprotein production due to the characteristics of the N-glycans in the expressed products. Insect cells lack several enzymes required for mammalian-type N-glycan synthesis and contain a specific N-acetylglucosaminidase that stunts the growth of chains and a core alpha-1,3-fucosyltransferase that yields potentially allergenic glycoforms. Current knowledge on N-glycan processing in lepidopteran insect cells is summarized, and strategies to develop better glycoprotein expression systems suitable for pharmaceutical glycoprotein production are discussed.

N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system.

N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection. N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were Man1-3(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%). There was only approximately 6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue. However alpha(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.

Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian [beta]-1,4-galactosyltransferase and [beta]-1,2-N-acetylglucosaminyltransferase II.

A novel recombinant baculovirus expression vector was used to produce His-tagged human transferrin in a transformed insect cell line (Tn5beta4GalT) that constitutively expresses a mammalian beta-1,4-galactosyltransferase. This virus encoded the His-tagged human transferrin protein in conventional fashion under the control of the very late polyhedrin promoter. In addition, to enhance the synthesis of galactosylated biantennary N-glycans, this virus encoded human beta-1,2- N-acetylglucosaminyltransferase II under the control of an immediate-early (ie1) promoter. Detailed analyses by MALDI-TOF MS, exoglycosidase digestion, and two-dimensional HPLC revealed that the N-glycans on the purified recombinant human transferrin produced by this virus-host system included four different fully galactosylated, biantennary, complex-type glycans. Thus, this study describes a novel baculovirus-host system, which can be used to produce a recombinant glycoprotein with fully galactosylated, biantennary N-glycans.