Bovine Pituitary Membrane Glycoproteins Contain β-N-Acetylgalactosaminylated N-Linked Sugar Chains

Abstract: Bovine pituitary glycoprotein hormones contain unique N-linked sugar chains with GalNAcβ1 → 4GlcNAc structure in their outer chain moieties. In the present study, whether bovine pituitary membrane glycoproteins contain the sugar chains with the disaccharide structure was investigated. Western blot analysis of the membrane glycoproteins using Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with β- N -acetylgalactosamine residue(s), showed that most protein bands detected with Coomassie Brilliant Blue staining bind to WFA. However, no WFA binding was observed for the bands after treatment of the blotted filter with jack bean β- N -acetylhexosaminidase or N -Glycanase. The WFA-positive bands were also detected in membrane glycoprotein samples from bovine cerebrum, cerebellum, and medulla oblongata, although their expression levels were low. Structural analysis of the oligosaccharides released by hydrazinolysis from the pituitary membrane glycoproteins by serial lectin column chromatography and sequential exoglycosidase digestion revealed that the major oligosaccharides, which bound to a WFA-agarose column, are of biantennary complex type with one and two GalNAcβ1 → 4GlcNAc groups in their outer chain moieties. These results indicate that the β- N -acetylgalactosaminylation is not unique to the glycoprotein hormones but occurs to most bovine pituitary glycoproteins.     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

Structural refinement of insecticidal plant proteinase inhibitors from Nicotiana alata

Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.     

A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata

A basic, galactose-rich style glycoprotein (GaRSGP) encoded by a previously characterized style-specific cDNA (NaPRP4) has been isolated from the styles of Nicotiana alata and structurally characterized. The glycoprotein is associated with cell walls in the transmitting tract and is composed of approximately 25% (w/w) protein and 75% (w/w) carbohydrate. The purified glycoprotein appears as a smear of between 45–120 kDa on SDS—PAGE; the deglycosylated protein backbone has an apparent molecular weight of approximately 30 kDa. The glycoprotein is rich in the amino acids lysine, proline, and hydroxyproline and in the monosaccharides galactose and arabinose. It is one of only a few proline/hydroxyproline-rich glycoproteins (P/HRGPs) to be characterized both as a cDNA-clone and protein. Glycans are attached to the protein backbone through both O - and N -glycosidic linkages with the majority of the carbohydrate being O -linked and consisting of short, highly branched chains terminating primarily in galactose residues. A carbohydrate epitope(s) is found on both GaRSGP and another style-specific glycoprotein but not on glycoproteins from other tissues. This finding provides further evidence for the existence of a style-specific carbohydrate epitope(s) which may play a role in style function.     

Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests

The ornamental tobacco (Nicotiana alata) produces one 6-kDa chymotrypsin inhibitor and four 6-kDa trypsin inhibitors from a single 40.3-kDa precursor protein. Three different approaches have been used to assess the potential of these proteinase inhibitors (PIs) in insect control. The first was an in-vitro approach in which all five inhibitors, the single chymotrypsin inhibitor or three of the four trypsin inhibitors were tested for their ability to inhibit gut protease activity in insects from four orders. The second approach was to incorporate the N. alata PIs in the artificial diet of the native budworm (Helicoverpa punctigera) and the black field cricket (Teleogryllus commodus). H. punctigera larvae and T. commodus nymphs had a significant (P<0.01) reduction in growth after ingestion of the PI and were more lethargic than insects on the control diet. Several of the H. punctigera larvae also failed to complete moulting at the third or fourth instar. The third approach was to express the N. alata PIs in transgenic tobacco under the control of the 35S CaMV promoter. When H. punctigera larvae were fed tobacco leaves expressing the N. alata PIs at 0.2% soluble protein, significant (P<0.01) differences in mortality and/or growth rate were observed.     

Exchanging sequence domains between S-RNases from Nicotiana alata disrupts pollen recognition

In self-incompatible plants of the Solanaceae, the specificity of pollen rejection is controlled by a single multiallelic S-locus. Pollen tube growth is inhibited in the style when its single S-allele matches either S-allele present in the diploid pistil. Each S-allele encodes an S-RNase with a unique sequence. S-RNases are secreted into the extracellular matrix of the transmitting tract which guides pollen tubes toward the ovary. Although it is known that S-RNases are the determinants of S-allele specificity in the pistil, it is not known how allele-specific information is encoded in the sequence. Therefore, we exchanged domains between S-RNases with different recognition specificities and expressed the chimeric proteins in transgenic plants to determine their effects on pollination behavior. Nine chimeric constructs were prepared in which domains from Nicotiana alata S_A2- and S_C10-RNases were exchanged. Among these nine constructs, the entire S-RNase sequence was sampled by exchanging single variable domains as well as larger blocks of contiguous sequences. The chimeric S-RNases retained enzymatic activity and were expressed at levels comparable to control transformants expressing S_A2- and S_C10-RNase. However, none of the chimeric S-RNases caused rejection of either S_A2- or S_C10-pollen. We conclude that the recognition function of S-RNases can be disrupted by alterations in many parts of the sequence. It appears that the recognition function of S-RNase is not localized to a specific domain.     

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc₃Man₉GlcNAc₂ precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-³H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man_9-8GlcNAc₂. In contrast, unstable ERAD substrates are trimmed to Man_6-5GlcNAc₂ and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.Download video file.^{(118M, mp4)}     

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.     

Sialylation of N-Carbohydrate Chains of Glycoproteins with Trans-Sialidase from Trypanosoma cruzi

2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. ruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for 2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.     

S-specific proteins in styles of self-incompatible Nicotiana alata

Summary A comparison of the stigma protein patterns of individual plants of the inbred- and cross-progenies in Nicotiana alata by isoelectric focusing revealed the presence of S-specific proteins. The S allele-protein relationship was found for three different S alleles. The S-specific proteins occurred in both stigma and stylar parts of the pistil whereas they were absent in leaves. In clone OWL the concentration of S-specific proteins in the stigma increased gradually during floral development. The shift from compatibility to incompatibility was not accompanied by an abrupt increase in concentration of the S-proteins.     

Modification of the Campylobacter jejuni N-Linked Glycan by EptC Protein-mediated Addition of Phosphoethanolamine

Campylobacter jejuni is the major worldwide cause of bacterial gastroenteritis. C. jejuni possesses an extensive repertoire of carbohydrate structures that decorate both protein and non-protein surface-exposed structures. An N-linked glycosylation system encoded by the pgl gene cluster mediates the synthesis of a rigidly conserved heptasaccharide that is attached to protein substrates or released as free oligosaccharide in the periplasm. Removal of N-glycosylation results in reduced virulence and impeded host cell attachment. Since the N-glycan is conserved, the N-glycosylation system is also an attractive option for glycoengineering recombinant vaccines in Escherichia coli. To determine whether non-canonical N-glycans are present in C. jejuni, we utilized high throughput glycoproteomics to characterize C. jejuni JHH1 and identified 93 glycosylation sites, including 34 not previously reported. Interrogation of these data allowed the identification of a phosphoethanolamine (pEtN)-modified variant of the N-glycan that was attached to multiple proteins. The pEtN moiety was attached to the terminal GalNAc of the canonical N-glycan. Deletion of the pEtN transferase eptC removed all evidence of the pEtN-glycan but did not globally influence protein reactivity to patient sera, whereas deletion of the pglB oligosaccharyltransferase significantly reduced reactivity. Transfer of eptC and the pgl gene cluster to E. coli confirmed the addition of the pEtN-glycan to a target C. jejuni protein. Significantly reduced, yet above background levels of pEtN-glycan were also observed in E. coli not expressing eptC, suggesting that endogenous E. coli pEtN transferases can mediate the addition of pEtN to N-glycans. The addition of pEtN must be considered in the context of glycoengineering and may alter C. jejuni glycan-mediated structure-function interactions.     

红曲霉葡萄糖淀粉酶N-连接糖链的初步研究

 在加强了的碱性条件下对红曲霉葡萄糖淀粉酶进行β-消除反应,分离未发生消除的级分。对反应前后的样品进行糖组成及甲基化分析,证实了红曲霉葡萄糖淀粉酶上确有对β-消除有抵抗的含GlcNAc的糖链存在。     

Effects of N-Linked Glycan on Lassa Virus Envelope Glycoprotein Cleavage,Infectivity, and Immune Response

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. The glycoprotein complex (GPC) contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage, transport, receptor recognition, epitope shielding, and immune response. We used three mutagenesis strategies (asparagine to glutamine, asparagine to alanine, and serine/tyrosine to alanine mutants) to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd (N89), 5th (N119), or 8th (N365) glycosylation motif. To evaluate N to Q mutagenesis for further research, it was found that deletion of the 2nd (N89Q) or 8th (N365Q) glycan completely inhibited the transduction efficiency of pseudotyped particles. We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice. Deletion of the individual 1st (N79Q), 3rd (N99Q), 5th (N119Q), or 6th (N167Q) glycan significantly enhanced the proportion of effector CD4+ cells, whereas deletion of the 1st (N79Q), 2nd (N89Q), 3rd (N99Q), 4th (N109Q), 5th (N119Q), 6th (N167Q), or 9th (N373Q) glycan enhanced the proportion of CD8+ effector T cells. Deletion of specific glycan improves the Th1-type immune response, and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC. However, the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV. The glycan residues on GPC provide an immune shield for the virus, and thus represent a target for the design and development of a vaccine.     

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

Properties of Two N-Linked Glycoproteins Associated with the Nuclear Envelope in Mung Bean Hypocotyls

Nuclei from mung bean (Vigna radiata) hypocotyls contained twoglycoproteins of 50 and 49 kDa, respectively, that reacted withconcanavalin A. The glycoproteins were released from the nuclearenvelope by treatment with 2 M KCl but not with nucleases. Theglycoproteins, tentatively named gp50 and gp49, were isolatedand characterized. Gel-permeation chromatography suggested thatgp50 and gp49 seem to exist as a complex with other components.The glycoproteins could be detected only in the nuclear fractionby immunoblot analysis with specific antibodies, and they werenot detected in endoplasmic reticulum, plasma membrane, vacuolarmembrane or mitochondria. Agglutinin I from Ulex europeaus,peanut agglutinin, soybean agglutinin and wheat germ agglutininall failed to bind to the glycoproteins. Treatment with glycopeptidaseF removed all oligosaccharides from the glycoproteins and decreasedtheir molecular masses by about one thousand daltons each. Theseresults suggest that the glycoproteins contained N-linked, high-mannose-typeoligosaccharides with six or seven hexose residues. gp50 andgp49 seemed to be isoforms of a single glycoprotein becausethe two proteins had some common properties. Nuclear fractionsfrom azuki bean (Phaseolus angularis) and soybean (Glycine max)contained proteins that were immunologically similar to gp50and gp49. (Received March 18, 1995; Accepted May 24, 1995)     

Nucleotide sequence and style-specific expression of a novel proline-rich protein gene from Nicotiana alata

cDNA clones encoding a novel proline-rich protein (NaPRP4) have been isolated from a Nicotiana alata stylar cDNA library. The N-terminal part of the derived protein is highly rich in proline (32.2%) and contains several repeats such as Lys-Pro-Pro (7 times) and Pro-Thr-Lys-Pro-Pro-Thr-Tyr-Ser-Pro-Ser-Lys-Pro-Pro (twice); the C-terminal part, on the other hand, has a lower proline content (9.9%) and contains two potential N-glycosylation sites and all the six cysteine residues. Northern blot and in situ hybridisation analyses indicate that expression of the NaPRP4 gene is restricted to cells of the transmitting tract of the style.     

Molecular and genetic characterization of novel S-RNases from a natural population of Nicotiana alata

Self-incompatibility in the Solanaceae is mediated by S-RNase alleles expressed in the style, which confer specificity for pollen recognition. Nicotiana alata has been successfully used as an experimental model to elucidate cellular and molecular aspects of S-RNase-based self-incompatibility in Solanaceae. However, S-RNase alleles of this species have not been surveyed from natural populations and consequently the S-haplotype diversity is poorly known. Here the molecular and functional characterization of seven S-RNase candidate sequences, identified from a natural population of N. alata, are reported. Six of these candidates, S ₅ , S ₂₇ , S ₇₀ , S ₇₅ , S ₁₀₇ , and S ₂₁₀ , showed plant-specific amplification in the natural population and style-specific expression, which increased gradually during bud maturation, consistent with the reported S-RNase expression. In contrast, the S ₆₃ ribonuclease was present in all plants examined and was ubiquitously expressed in different organs and bud developmental stages. Genetic segregation analysis demonstrated that S ₂₇ , S ₇₀ , S ₇₅ , S ₁₀₇ , and S ₂₁₀ alleles were fully functional novel S-RNases, while S ₅ and S ₆₃ resulted to be non-S-RNases, although with a clearly distinct pattern of expression. These results reveal the importance of performing functional analysis in studies of S-RNase allelic diversity. Comparative phylogenetic analysis of six species of Solanaceae showed that N. alata S-RNases were included in eight transgeneric S-lineages. Phylogenetic pattern obtained from the inclusion of the novel S-RNase alleles confirms that N. alata represents a broad sample of the allelic variation at the S-locus of the Solanaceae.