Oligosaccharides modulate the apoptotic activity of glycodelin

GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.     

Glycodelin: a reproduction-related lipocalin

Glycodelin, a human lipocalin, is a major endometrial protein with at least two differentially glycosylated isoforms. Glycodelin-A (GdA) is purified from human mid-trimester amniotic fluid, where it is secreted from the decidualized endometrium. Glycodelin-S (GdS) is synthesized in the male reproductive tract, mainly in the seminal vesicles, and secreted into seminal plasma. These two glycodelin isoforms, glycosylated in a completely different manner, serve as a good model for studying the effects of glycosylation on protein function and physicochemical properties. We have reviewed here the structure, expression and biological functions of glycodelin.     

Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA),⁴ glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2–5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complex-type structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc), NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc), NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc), Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3–5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5).Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10). For example, both GdA and GdF inhibit the spermatozoa-zona pellucida binding (11) via fucosyltransferase-5 (12), but only the latter inhibits progesterone-induced acrosome reaction, thus preventing a premature acrosome reaction of the spermatozoa. There is evidence that cumulus cells can convert exogenous GdA and -F to GdC, the physicochemical properties of which suggest that it is differently glycosylated compared with GdA/F (5). Moreover, GdC stimulated spermatozoa-zona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound GdA and -F (4, 5). GdS suppresses capacitation probably via its inhibitory activity on cholesterol efflux from spermatozoa (13).Except for the effects on fertilization, GdA is involved in fetomaternal defense. This glycodelin isoform suppresses proliferation and induces apoptosis of T cells (2) and inhibits natural killer cell (14) and B-cell (15) activities. Glycosylation is involved in the binding of GdA to receptors on T cells (16). The sialic acid of GdA contributes to the apoptotic activity in T cells (17, 18) and binding to CD45, a potential GdA receptor (16). The importance of glycosylation in glycodelin is further shown by the absence of immunosuppressive activities in GdS with different glycosylation (18). The immunomodulating activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different glycodelins to exhibit their binding activities and biological effects (13, 19, 20). The present study aims to identify the effect of all four glycodelin isoforms on lymphocyte viability, cell death, and interleukin-2 (IL-2) secretion and to correlate these bioactivities with their glycosylation patterns determined by mass spectrometry.     

Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cells but not in chinese hamster ovary cells.

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.     

C-reactive protein reduces protein S-nitrosylation in endothelial cells

Glycodelin A (GdA) is a dimeric glycoprotein synthesized by the human endometrium under progesterone regulation. Based on the high sequence similarity with β-lactoglobulin, it is placed under the lipocalin superfamily. The protein is one of the local immunomodulators present at the feto-maternal interface which affects both the innate as well as the acquired arms of the immune system, thereby bringing about successful establishment and progression of pregnancy. Our previous studies revealed that the domain responsible for the immunosuppressive activity of glycodelin lies on its protein backbone and the glycans modulate the same. This study attempts to further delineate the apoptosis inducing region of GdA. Our results demonstrate that the stretch of amino acid sequence between Met24 to Leu105 is necessary and sufficient to inhibit proliferation of T cells and induce apoptosis in them. Further, within this region the key residues involved in harboring the activity were shown to be present between Asp52 and Ser65.     

Analysis of the role of oligosaccharides in the apoptotic activity of glycodelin A

Glycodelin A, also known as placental protein-14, is a multifunctional glycosylated protein secreted by the uterine endometrium during the early phases of pregnancy. It is a known suppressor of T cell proliferation, inducer of T cell apoptosis, and inhibitor of sperm zona binding. Unlike in contraceptive activity, where the glycans on the molecule have been shown to play a crucial role, mutagenesis of the asparagines at sites of N-linked glycosylation (Asn(28) and Asn(63)) to glutamine shows that the apoptogenic activity of glycodelin A is executed by the protein backbone. Glycosylation at Asn(28) appears to play a role in the extracellular secretion of the molecule, as mutation of Asn(28) resulted in a significant decrease in the amount of secreted protein, and loss of both glycosylation sites reduced the secretion drastically. Our results also suggest that the loss of glycosylation does not affect the dimerization status of the molecule.     

Differential sialylation regulates the apoptotic activity of glycodelin A

Glycodelin A (GdA), a dimeric lipocalin, expressed by the uterine endometrium, is an immunomodulatory agent and induces apoptosis in T-cells. In this study we demonstrate that two populations of GdA with subtle differences in their net ionic charge are present in the amniotic fluid and that, apoptotic activity is exhibited only by the population with more sialic acid residues. Significantly, removal of sialic acid residues from the active populations of GdA abrogates the activity of the molecule, suggesting that the extent of sialylation might be a factor regulating the activity of GdA.     

Glycodelin A, an immunomodulatory protein in the endometrium, inhibits proliferation and induces apoptosis in monocytic cells

Glycodelin A (GdA), is a lipocalin with an immunomodulatory role, secreted by the endometrium under progesterone regulation and proposed to play a role in protecting the fetus from maternal immune attack. Glycodelin A has an inhibitory effect on the proliferation of T cells and B cells and also on the activity of natural killer cells. We have earlier demonstrated that the inhibitory effect of glycodelin A on T cell proliferation is due to apoptosis induced in these cells through the caspase-dependent intrinsic mitochondrial pathway. Studies reported until now have shown that glycodelin modulates the adaptive immune responses. We, therefore, decided to look at its effect, if any, on the innate immune system. The effect of glycodelin on monocytes was studied using human monocytic cell lines, THP1 and U937, and primary human monocytes as model systems. We demonstrated that glycodelin inhibited the proliferation of THP1 and U937 and induced apoptosis in these cells as well as in primary monocytes. We found that this signaling was caspase-independent but followed the intrinsic mitochondrial pathway of apoptosis. No effect of glycodelin was seen on the phagocytic ability of monocytes post-differentiation into macrophages. These observations suggest that, at the fetomaternal interface, glycodelin plays a protective role by deleting the monocytes that could become pro-inflammatory. Importantly, leaving the macrophages untouched to carry on with efficient clearance of the apoptotic cells.     

Structures of Merkel Cell Polyomavirus VP1 Complexes Define a Sialic Acid Binding Site Required for Infection

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.     

Effect of pH on the structure and aggregation of human glycodelin A. A comparison with beta-lactoglobulin A

The effect of pH on the structure of glycodelin A (GdA) and of beta-lactoglobulin A (beta-LgA) has been investigated by means of circular dichroism, steady state fluorescence, synchrotron radiation small angle X-ray scattering (SR-SAXS) and gel permeation chromatography. The comparison between GdA and beta-LgA shows that, at pH 7.0, both proteins are dimers with an extended content of beta-sheet conformation, but pH 2.0 and 9.0 yield a different secondary, tertiary and quaternary structural organisation. Whilst beta-LgA is a monomer, that conserves beta-sheet conformation at pH 2.0 and 9.0, GdA has a stable dimeric structure at alkaline pH, but at pH 2.0 increases its alpha-helix content and it aggregates soon. SR beam has been used to perform SAXS comparative measurements of the two proteins. SR-SAXS data provide the radius of gyration and the radii of the cross-section and of the thickness. GdA aggregation at acid pH has been characterised by calculating the distance distribution function (P(r)). Isoelectric focusing and chromatofocusing data show a different charge distribution on the surfaces of the two proteins, supporting the hypothesis that the presence of oligosaccharides deeply influences the conformational state and the aggregation process of GdA at different pH values. In particular, the presence of sialic acid residues, within the oligosaccharide moiety of the GdA, might be responsible for the differences observed between the two proteins.     

Structural properties of human glycodelin A in water and in water-alcohol mixtures: a comparison with bovine beta-lactoglobulin A.

Human glycodelin A (GdA) is a glycoprotein that is highly homologous to bovine beta-lactoglobulin A (beta-LgA) because the amino acid sequences are 50-60% identical. The structural characteristics of human GdA and beta-LgA were compared in water and 2-propanol/water solutions. Circular dichroism spectra reveal that in water the two proteins have a very similar beta-sheet secondary structure. In the presence of 2-propanol/water mixtures (up to 50% v/v) the alpha-helix structure of both proteins increases. A further increase in the alcohol percentage of the solvent (up to 80% v/v 2-propanol) causes the formation of a new folded tertiary structure containing mainly beta-sheet features. Synchrotron radiation small angle X-ray scattering indicates that, in a neutral pH aqueous solution, GdA is a dimer. Its radius of gyration value (Rg), 25.1+/-0.4 A, is greater than that of beta-LgA (21.1+/-0.3 A), probably because of the contribution of polysaccharides bound to Asn-28 and Asn-63 residues of GdA. Conversely, small angle X-ray scattering and gel permeation chromatography data on GdA in 2-propanol have revealed a massive aggregation of the protein.     

Glycodelin-A Stimulates Interleukin-6 Secretion by Human Monocytes and Macrophages through L-selectin and the Extracellular Signal-regulated Kinase Pathway

Macrophages represent the second major type of decidual leukocytes at the fetomaternal interface. Changes in macrophage number and activity are associated with fetal loss and pregnancy complications. Glycodelin-A (GdA) is an abundant glycoprotein in the first-trimester decidua. It is involved in fetomaternal defense and early placental development through its regulatory activities in various immune cells. The N-glycosylation of GdA mediates the binding and therefore the activities of the molecule. In this study, we studied the biological activities of GdA in the functions of human monocytes/macrophages. GdA was purified from amniotic fluid by affinity chromatography. GdA treatment did not affect the viability, cell death, or phagocytic activity of the monocytes/macrophages. GdA, but not recombinant glycodelin without glycosylation, induced IL-6 production as demonstrated by cytokine array, intracellular staining, and ELISA. GdA also induced phosphorylation of ERK in monocytes/macrophages. The involvement of ERKs in IL-6 induction was confirmed using pharmacological inhibitors. Co-immunoprecipitation showed that L-selectin on the monocytes/macrophages was the binding protein of GdA. Treatment with anti-L-selectin antibody reduced GdA binding and GdA-induced IL-6 production. GdA-treated macrophages suppressed IFN-γ expression by co-cultured T-helper cells in an IL-6-dependent manner. These results show that GdA interacts with L-selectin to induce IL-6 production in monocytes/macrophages by activating the ERK signaling pathway. In turn, the increased IL-6 production suppresses IFN-γ expression in T-helper cells, which may play an important role in inducing a Th-2-polarized cytokine environment that flavors the immunotolerance of the fetoplacental unit.     

Engineering host cell lines to reduce terminal sialylation of secreted antibodies

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.Key words: antibodies, IgGs, glycans, oligosaccharides, sialic acid, sialidase, ADCC, CDC, effector functions, cells, Fc receptors, proteases     

Membrane biogenesis in embryonal carcinomas: glycoproteins destined for the cell surface are delayed in a pre-Golgi compartment

Embryonal carcinoma and early embryonic cells assemble a family of unusually large and complex carbohydrates. These glycans are highly branched, repeating copolymers of the sugars galactose and N-acetylglucosamine, referred to as polylactosamines, and are frequently decorated with fucose, sulfate, and sialic acid. We have previously shown that in teratocarcinoma cells these glycans are part of a large spectrum of glycans assembled on mannose cores derived from a common precursor glycan. Metabolic studies revealed a large excess of high-mannose glycans at a time when complex-type glycans cease to accumulate. The present studies demonstrate that these high-Man glycans are not degraded internally or secreted directly but are on glycoproteins destined for the cell surface. These unprocessed glycoproteins replace material lost during the extensive membrane turnover that occurs in these cells. Their export to the cell surface is delayed in a pre-Golgi compartment.     

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.     

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) secrete a number of acid hydrolases and other proteins that have both highmannose and complex asparagine-linked glycans. We used affinity chromatography with concanavalin A and an antiserum specific for complex glycans in conjunction with in vivo-labeling studies to show that all of the secreted proteins carry glycans. The presence of complex glycans on secretory proteins indicates that they are passing through the Golgi complex on the way to the extracellular compartment. The sodium ionophore, monensin, did not block the transport of proteins to the extracellular medium, even though monensin efficiently inhibited the Golgi-mediated processing of complex glycans. The inhibition of N-glycosylation by tunicamycin reduced by 76% to 84% the accumulation of newly synthesized (i.e. radioactively labeled) protein that was secreted by the sycamore cells, while cytoplasmic protein biosynthesis was not affected by this antibiotic. However, in the presence of glycoprotein-processing inhibitors, such as castanospermine and deoxymannojirimycin, the formation of complex glycans was prevented but glycoprotein secretion was unchanged. These results support the conclusion that N-linked glycan processing is not necessary for sorting, but glycosylation is required for accumulation of secreted proteins in the extracellular compartment.     

Immunolocalization of glycodelin in the genital tract of rats

Glycodelin, also known as placental protein 14 has been predominantly localized to organs of the human genital tract. Unfortunately the physiological role of glycodelin is largely unknown since it depends on limited availability of tissues. Therefore, a suitable animal model to study the role of glycodelin would be desirable. Previously, it was shown that glycodelin mRNA is expressed in the genital tract of male and female rats. In the present study, we demonstrate the expression of glycodelin protein in male and female rats by immunohistochemistry and Western blot analysis. For this purpose a polyclonal antibody was generated against glycodelin peptide. In female rats, glycodelin was found in the epithelial gland cells of the uterus, epithelial cells of the fallopian tube as well as in corpora lutea, interstitial and theca cells of the ovary. Glycodelin was distributed in all epithelial cells of the epididymis and the seminal vesicle. In the seminiferous epithelium, glycodelin was seen in all developmental stages of spermatogonia and spermatocytes and in Sertoli cells. Whereas in the rat male reproductive tract glycodelin expression is slightly different from human or primate tissues, in organs of the rat female genital tract glycodelin expression is similar to humans and primates.     

Glycosylation influences the lectin activities of the macrophage mannose receptor

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man5-GlcNAc2 glycans (Man5 -MR) or non-sialylated complex glycans (SA0-MR) did not bind mannosylated glycans, but could recognize SO4-3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA0-MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands.     

Expression and characterization of human N-acetylglucosaminyltransferases and alpha2,3-sialyltransferase in insect cells for in vitro glycosylation of recombinant erythropoietin

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennary-type complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of N-acetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using UDP-14C-Gal and CMP-14C-Sia as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the red blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.     

N-glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures.

Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.