N-linked Carbohydrate Residues Research Articles

Enhanced Zika virus-like particle development using Baculovirus spp. constructs.

Zika virus (ZIKV) is one of several examples of an unprecedented pandemic spread and against which there is currently no suitable vaccine or treatment.Here, we constructed and characterized recombinant baculovirus-derived ZIKV-like particles (Zika VLPs) to study ZIKV-antibody interactions. These VLPs, uniquely consisted of the full-length ZIKV capsid (C), pre-membrane (prM), and envelope (E) proteins with either: a) the viral nonstructural NS2B and NS3 protease unit under one or two different promoters or b) an alternative host-cell furin protease encoding cleavage sequence inserted between the C and prM genes, together with lobster tropomyosin leader and honeybee signal sequences with one promoter for increased extracellular secretion.All these Zika VLPs displayed typical virion morphology in transmission electron microscopicanalysis when expressed in both insect (Sf9) and mammalian (HEK293T) cells and no uncleaved prM glycoprotein was detected, as are present on immature virions. The importance of glycosylation of the E glycoprotein was shown by the effects on both polyclonal and monoclonal antibody reactions after these N-linked carbohydrate residues were disrupted by oxidation or enzymatic cleavage.Importantly, the construct which contained the host-cell furin protease cleavage sequence together with a lobster tropomyosin leader and honeybee signal sequences under one promoter produced higher Zika VLP titers and protein concentrations and which can now be tested as a superior construct in multifunctional diagnostic (ELISA and neutralization/antibody-dependent enhancement) assays and immunogenic assessments possibly leading to vaccine trials.

Site-specific roles of N-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction

Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific β-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH β-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn22 (eelFSHβΔ22/α) and Asn5.22 (eelFSHβΔ5.22/α) N-linked oligosaccharide chain in the eelFSH β-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHβ/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHβ/α wild-type by western blotting analysis was found to be 34 kDa. Mutants (β/αΔ56, β/αΔ79, and βΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHβ/α wild-type were modified to a molecular weight of approximately 7–10 kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn56 and Asn79 on the α-subunit and Asn5 on the β-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC50 values of Asn56 and Asn5 resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn79 of the α-subunit (β/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.

Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface.

Collagen XVII/BP180, an epithelial adhesion molecule, belongs to the group of collagenous transmembrane proteins, which are characterized by ectodomain shedding. We recently showed that ADAMs can cleave collagen XVII, but also that furin participates in this process (Franzke, C. W., Tasanen, K., Schäcke, H., Zhou, Z., Tryggvason, K., Mauch, C., Zigrino, P., Sunnarborg, S., Lee, D. C., Fahrenholz, F., and Bruckner-Tuderman, L. (2002) EMBO J. 21, 5026-5035). To define the cleavage region in the juxtamembranous NC16A linker domain and assess its structure and requirements for shedding, we constructed deletion mutants of the NC16A domain, expressed them in COS-7 cells, and analyzed their structural integrity and shedding behavior. A mutant lacking the furin consensus sequence was shed in a normal manner, demonstrating that furin does not cleave collagen XVII but rather activates ADAMs (a disintegrin and metalloproteinase). Large deletions of the NC16A domain prevented shedding, and analysis of defined smaller deletions pointed to the stretch of amino acid residues 528-547 as important for sheddase recognition and cleavage. Secondary protein structure predictions showed that deletion of this stretch resulted in an NC16A domain with a positive net charge and an amphipathic alpha-helix, which can cause conformational changes in the collagen XVII homotrimer. Assessment of triple-helix folding of the mutants revealed a lower thermal stability of all non-shed variants than of wild-type collagen XVII or the shed mutants. In contrast, deletion of the putative nucleation site for triple-helix folding of collagenous transmembrane proteins did not affect folding of collagen XVII. The data indicate that the conformation of the NC16A domain and steric availability of the cleavage site influence shedding and is important for folding of collagen XVII.

A product of DAN, a novel candidate tumour suppressor gene, is secreted into culture medium and suppresses DNA synthesis.

Our previous studies have shown that the DAN gene product possesses an ability to revert phenotypes of transformed rat fibroblasts and represents a candidate tumour suppressor gene for neuroblastoma. In the present study, characterisation of DAN was carried out using rat fibroblast 3Y1 cells and their DAN-overexpressor counterparts (S-9). The N-terminal region of DAN (amino acids 1-24) was highly hydrophobic and DAN protein was found to be secreted into the culture medium. When DAN was treated with PNGase F, a enzyme that cleaves most N-linked carbohydrate residues, the mobility of both cytoplasmic and secreted DAN was increased in SDS-polyacrylamide gel electrophoresis, suggesting DAN is N-glycosylated, irrespective of its localisation. When partially purified, DAN was able, when added to the culture, to suppress DNA synthesis of Rous sarcoma virus-transformed 3Y1 cells, which lack the expression of DAN.

Characterization of platelet aggregation induced by the human carcinosarcoma Colo 526: role of platelet activation, tumor cell cytoskeleton and tumor cell plasma membrane

Tumor cell-platelet interactions have been shown to be involved in the process of metastasis. This study characterizes the aggregation of washed platelets induced by the human uterine carcinosarcoma Colo 526. Ultrastructural studies revealed a two-stage process in which the earliest events were the adhesion and degranulation of individual platelets in contact with the tumor cell membrane. The second stage consisted of a wave of aggregation involving all residual platelets. We found that the first stage was initiated by a factor integral to the tumor cell plasma membrane which acted independently of the tumor cell cytoskeleton or metabolic processes. This factor was found to be a glycoprotein or glycolipid with functionally important sialic acid and N-linked carbohydrate residues. The initial stage was not dependent on platelet activation as neither aspirin nor prostacyclin prevented adhesion or degranulation. The second stage was found to be dependent on platelet activation. These results suggest that platelet aggregation induced by Colo 526 involves a distinctive primary stage which is initiated by a factor on the tumor cell plasma membrane resulting in the degranulation and lysis of individual platelets. This process can occur independently of platelet activation or aggregation and thus may have some relevance to the clinical use of platelet antagonists as antimetastatic agents.

L273S missense substitution in human lysosomal acid lipase creates a new N-glycosylation site

Human lysosomal acid lipase (LAL), when expressed in Hela cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglycosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to N-glycosylation. A missense substitution, L273S, previously detected in a patient with cholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS → NML) indicated that the L273S mutation creates an additional N-glycosylation consensus (N-X-S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglycosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N-glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri-oleyl glycerol and cholesteryl oleate by 50%, strongly suggesting that N-linked carbohydrate residues are important for optimal catalytic activity.

Lymphotoxin‐α/β heterodimer is expressed on leukemic hairy cells and activated human B lymphocytes

The expression of human lymphotoxin (LT) alpha/beta cell-surface complex was studied in human B-cell lines as well as in normal and neoplastic human B lymphocytes. In the absence of TNF receptors, only the human hairy-cell leukemia (HCL)-derived cell line JOK-I revealed constitutive cell-surface expression of LT but not TNF-alpha. Immunoprecipitation experiments with anti-LT monoclonal antibody (MAb) 9B9 from cell-surface radioiodinated JOK-I cells revealed that a cell-surface lymphotoxin molecule (25 kDa) is expressed in association with a 33-kDa molecule. Enzymatic digestion with F/N-glycosidase and O-glycosidase showed that both proteins contained N-linked carbohydrate residues, whereas only the 25-kDa molecule contained O-linked sugar residues. Analysis of mRNA expression revealed specific transcripts of LT-alpha and LT-beta in JOK-I cells. Resting tonsillar B cells did not express cell-surface LT. However, LT-beta mRNA was observable in unstimulated tonsillar B cells, whereas LT-alpha mRNA, cell-surface LT and LT secretion could only be detected upon in vitro activation. Thus LT-beta and alpha appear to be sequentially expressed in human B cells. Neoplastic B cells from chronic lymphocytic leukemia (BCLL), being devoid of constitutive cell-surface LT expression, could be induced to express surface LT by in vitro stimulation with Staphylococcus aureus Cowan I (SAC). Constitutive LT-beta transcripts, however, could also be detected in 4 out of 5 cases of BCLL. In contrast, human HCL cells displayed constitutive cell expression of lymphotoxin-alpha and beta. These findings demonstrate that cell-surface LT-alpha is expressed in association with LT-beta on activated normal B cells and neoplastic B cells representing an activated state.

Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3.

To analyze the binding requirements of LFA-1 for its two most homologous ligands, ICAM-1 and ICAM-3, we compared the effects of various LFA-1 activation regimes and a panel of anti-LFA-1 mAbs in T cell binding assays to ICAM-1 or ICAM-3 coated on plastic. These studies demonstrated that T cell binding to ICAM-3 was inducible both from the exterior of the cell by Mn2+ and from the interior by an agonist of the "inside-out" signaling pathway. T cells bound both ICAM ligands with comparable avidity. A screen of 29 anti-LFA-1 mAbs led to the identification of two mAbs specific for the alpha subunit of LFA-1 which selectively blocked adhesion of T cells to ICAM-3 but not ICAM-1. These two mAbs, YTH81.5 and 122.2A5, exhibited identical blocking properties in a more defined adhesion assay using LFA-1 transfected COS cells binding to immobilized ligand. Blocking was not due to a steric interference between anti-LFA-1 mAbs and N-linked carbohydrate residues present on ICAM-3 but not ICAM-1. The epitopes of mAbs YTH81.5 and 122.2A5 were shown to map to the I domain of the LFA-1 alpha subunit. A third I domain mAb, MEM-83, has been previously reported to uniquely activate LFA-1 to bind ICAM-1 (Landis, R. C., R. I. Bennett, and N. Hogg. 1993. J. Cell Biol. 120:1519-1527). We now show that mAb MEM-83 is not able to stimulate binding of T cells to ICAM-3 over a wide concentration range. Failure to induce ICAM-3 binding by mAb MEM-83 was not due to a blockade of the ICAM-3 binding site on LFA-1. This study has demonstrated that two sets of functionally distinct mAbs recognizing epitopes in the I domain of LFA-1 are able to exert differential effects on the binding of LFA-1 to its ligands ICAM-1, and ICAM-3. These results suggest for the first time that LFA-1 is capable of binding these two highly homologous ligands in a selective manner and that the I domain plays a role in this process.

The Role of N-Linked Carbohydrate Residues in Lymphokine-Activated Killer Cell-Mediated Cytolysis

We evaluated LAK cell cytotoxicity toward a sensitive B cell lymphoma and several resistant EBV-transformed cell lines in order to explore the mechanism by which some cells are preferentially recognized as targets. Cytolysis of the sensitive cells was inhibited by monoclonal antibodies against the surface proteins LFA-1 and ICAM-1; however, surface expression of ICAM-1 was similar on the resistant and sensitive cell lines. Prevention of post-translational addition of N-linked oligosaccharides by treatment of the resistant cells with tunicamycin resulted in a dramatic enhancement in LAK cell cytotoxicity which was partially inhibited by antibodies against LFA-1 and ICAM-1. Treatment of the resistant cells with the endoglycosidase N-glycanase also increased LAK cell sensitivity. Tunicamycin treatment caused a decrease in the molecular weight of ICAM-1 from approximately 95,000 to 50,000 Da. Conjugate formation between the LAK cells and the sensitive and resistant target cells was similar before and after deglycosylation. We conclude that carbohydrate modification of ICAM-1 or an alternative glycoprotein confers resistance to LAK cell cytotoxicity in some cell lines.

Formation of A hCG variant with truncated peptide moiety as a biosynthetic product in vitro

Summary In serum of tumor patients variants of human chorionic gonadotropin (hCG) or of the free β-subunit with lower molecular weights may be present as well as proteolytically nicked forms. The origin of these variants, secreted by cells or formed in the periphery, is unknown. In the present study, brefeldin A (BFA) was used to block transiently the anterograde transport from the ER to the Golgi apparatus in JAr cells to increase the residency of hCG in the ER and thus to amplify possible proteolytic processes taking place in this cellular compartment. In JAr cells, BFA (2 hours, 0.05-0.4 μg/ml) diminished the amount of secreted free β-subunit (up to 90%) in a dose-dependent and reversible manner; hCG secretion was less affected. In the BFA cultures, the molar ratio hCG/free was increased up to five-fold. Pulse-chase experiments, using [ 3 H]-Leu for labeling of newly synthesized protein, showed the presence of lower molecular weight variants of the hCG-α and β subunits when hCG was arrested in the ER in presence of BFA. This could be observed most clearly in 8-bromo-cAMP (1 mM) stimulated cultures. In BFA treated cultures, the free β-subunit as well as the β-subunit contained in hCG showed and about 3 kD lower apparent molecular weight in comparison to the β-subunits of controls (34 kD). The lower molecular weight variants of the β-subunits still contained the carboxy-terminal peptide (CTP) since they were bound to anti-CTP-Sepharose. The carbohydrate part could also not account for the difference in the apparent molecular weight since it was not abolished by removal of the N-linked carbohydrate residues. The results demonstrate the capability of the ER to produce truncated variants of the free β-subunit and a truncated β-subunit contained in hCG which are secreted in vitro . During a transient arrest of the de novo synthesized proteins in the ER, the free β-subunit as well as hCG may be most probably proteolytic cleaved in the amino-terminal domain of the β-subunit or in the core part of the molecule.

Polylactosaminoglycan modification of the respiratory syncytial virus small hydrophobic (SH) protein: A conserved feature among human and bovine respiratory syncytial viruses

We investigated the nature of the oligosaccharide modification of the glycosylated forms of the small hydrophobic integral membrane protein, SH (previously designated 1A), of respiratory syncytial (RS) virus. Analysis of SH protein expressed in cells infected with RS virus or with a recombinant vaccinia virus revealed two glycosylated SH protein species, SH 9 and SH p, which contained N-linked carbohydrate residues. SH p migrated diffusely on polyacrylamide gels, which suggested modification by polylactosaminoglycan oligosaccharides. Polylactosaminoglycan modification of SH p was established from three lines of investigation: (1) the synthesis of SH p in a cell line (IdID) conditionally defective in the ability to add specific carbohydrate residues to N- or O-linked oligosaccharide chains required the addition of galactose, which is a component of the N-acetyllactosamine repeating unit; (2) SH p was sensitive to digestion with endo-,β-galactosidase, which cleaves the β1–4 linkage between galactose and N-acetylglucosamine of the repeated N-acetyllactosamine subunit; and (3) SH p was selected by Datura stramonium lectin (Dsl), which has specificity for polylactosaminoglycans. The presence of SH p as a component of purified human subgroups A and B and bovine RS virus particles was demonstrated by Dsl affinity selection. In addition to SH p, nonglycosylated SH 0 was selected by Dsl affinity, indicating that SH p and SH 0 may associate to form complexes within infected cells and virus particles. To identify conserved amino acid residues among the human and bovine SH glycoproteins that may function as signals for polylactosaminoglycan modification, the nucleotide sequences of the SH protein genes of a human subgroup B virus ( 8 60 ) and a bovine virus (391-2) were determined and compared to those of a human subgroup A virus (A2), a subgroup B virus (18537), and a bovine virus (A51908). A comparison of the deduced amino acid sequences of the human and bovine RS virus SH proteins indicated that a central hydrophobic region and the presence of potential N-linked glycosylation sites on either side of the central hydrophobic region were conserved features that may be required for the polylactosaminoglycan modification of SH.

Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM).

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.

Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes.

CD16 Ag is associated with the low affinity FcR for IgG expressed on human NK cells and granulocytes. In this study, we demonstrate that NK cells specifically lyse murine anti-CD16 hybridoma cell lines, but do not lyse hybridomas against other cell surface differentiation Ag expressed on NK cells. Moreover, the CD18 structure is involved in the CD16-specific xenogeneic interaction between human effector cells and murine hybridoma target cells. Although interaction with anti-CD16 hybridomas or antibodies triggers the cytolytic mechanism of NK cells, this interaction does not induce cellular proliferation. In contrast to NK cells, CD16+ granulocytes do not lyse anti-CD16 hybridoma cell targets and do not mediate ADCC against antibody-coated human tumor cell targets. These findings indicate a fundamental difference in the antibody-dependent cellular cytotoxicity mechanisms of NK cells and granulocytes. Comparative biochemical analysis of CD16 on NK cells and granulocytes revealed significant differences in the size of the polypeptides obtained after removal of N-linked carbohydrate residues with endo-F and N-glycanase digestion.

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains.

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.

Epithelium-specific surface glycoprotein of Mr 34,000 is a widely distributed human carcinoma marker

An epithelial cell surface antigen is described which is defined by monoclonal antibody HEA125 (IgG1). The antibody was raised against the colon carcinoma cell line HT-29. Under reducing conditions HEA125 immunoprecipitates a surface glycoprotein of Mr 34,000 which was designated Egp34. The antigen does not contain disulfide-linked subunits. A slightly different migration behavior under non-reducing conditions (Mr 39,000) may be due to intrachain disulfide bonds. After enzymatic cleavage of N-linked carbohydrate residues the apparent molecular weight of the antigen was 29,000. Egp34 is a major cell surface component of HT-29 cells (10(6) molecules per cell). No antigen could be detected in the sera of colorectal cancer patients. A panel of malignant cell lines and normal cells was studied for surface expression of the antigen. 17/17 carcinoma lines of 6 different origins expressed the antigen, whereas 16/16 melanoma, neuroblastoma, sarcoma and lymphoma/leukaemia were unreactive as it was the case for normal fibroblasts and blood cells. Immunoperoxidase staining of frozen tissue sections with HEA125 demonstrated the presence of Egp34 in almost all normal epithelia and tumours derived therefrom. No reactivity with non-epithelial tissues was observed. Undifferentiated carcinomas of various origins homogeneously expressed Egp34. Therefore, HEA125 may become a valuable tool for the immunohistochemical diagnosis of carcinoma.

Functional properties of carbohydrate-depleted tissue plasminogen activator.

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.