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1.
We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.  相似文献   

2.
Epidermal Growth Factor Receptor (EGFR) is a glycosylated tyrosine kinase receptor associated with several cancers. EGFR plays an important role in cancer therapy and inspired several experimental and computational (molecular dynamics simulation) studies to investigate its function and dynamics. N-glycosylation is a critical aspect of EGFR functioning that was mainly unexplained until recently due to the challenges in obtaining and analysis of the structural data involving the glycan moieties. Latest simulations of glycosylated EGFR suggest atomistic mechanisms underlying the experimentally proposed functions of N-glycans in: EGFR increased ligand binding, reduced flexibility and arrangement within the cell membrane. It was shown that the increase in the ligand binding of glycosylated EGFR is mediated by the interaction between the two glycans attached to the growth factor binding subdomains resulting in stabilization of the growth factor binding site. Persistent hydrogen bonds’ formation between the glycans and EGFR contributes to proper folding and reduced flexibly of the glycosylated receptor. Assembly of the cell-integrated EGFR and its relative distance from the membrane are acquired by the lift-up action of the attached glycans. These findings can be used as a framework for implementation of computational techniques to obtain atomistic details of protein glycosylation as one of the most important areas of structural biology.  相似文献   

3.
The structure and post-translational processing of the SARS-CoV-2 spike glycoprotein (S) is intimately associated with the function of the virus and of sterilising vaccines. The surface of the S protein is extensively modified by glycans, and their biosynthesis is driven by both the wider cellular context, and importantly, the underlining protein structure and local glycan density. Comparison of virally derived S protein with both recombinantly derived and adenovirally induced proteins, reveal hotspots of protein-directed glycosylation that drive conserved glycosylation motifs. Molecular dynamics simulations revealed that, while the S surface is extensively shielded by N-glycans, it presents regions vulnerable to neutralising antibodies. Furthermore, glycans have been shown to influence the accessibility of the receptor binding domain and the binding to the cellular receptor. The emerging picture is one of unifying, principles of S protein glycosylation and an intimate role of glycosylation in immunogen structure and efficacy.  相似文献   

4.
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.  相似文献   

5.
Follicle stimulating hormone (FSH) is one of the important hormones that regulate gonadal functions. This hormone is glycosylated, and the glycans greatly influence the biological properties. In the present study the negatively charged glycopeptides of equine and human pituitary follicle stimulating hormone (eFSH and hFSH) have been characterized in a glycosylation site-specific manner using FT-ICR-MS and Edman sequencing. The characteristic pattern of glycan distribution at each glycosylation site has been deduced and compared between horse and human FSH preparations. The data suggest that site-specific differences exist between glycoforms of human and equine FSH. For instance, except for one site in the beta subunit (Asn7) of hFSH all other sites in both species have sulfated glycoforms. Also, glycoforms at Asn52 of hFSH are all complex type, whereas in eFSH, both complex and hybrid structures exist at this site. There is also a higher percentage of sulfated glycans in the latter site compared to the former. This is the first study that characterizes the glycans from this hormone in a glycosylation site-specific manner, and these data can be used to begin correlative studies between glycosylation structure and hormone function.  相似文献   

6.
Tobacco-based transient expression was employed to elucidate the impact of differential targeting to subcellular compartments on activity and quality of gastric lipase as a model for the production of recombinant glycoproteins in plants. Overall N-linked glycan structures of recombinant lipase were analyzed and for the first time sugar structures of its four individual N-glycosylation sites were determined in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on a trypsin digest without isolation or deglycosylation of the peptides. Three glycosylation sites contain both complex-type N-glycans and high-mannose-type structures, the fourth is exclusively linked to high-mannose glycans. Although the overall pattern of glycan structures is influenced by the targeting, our results show that the type of glycans found linked to a given Asn residue is largely influenced by the physico-chemical environment of the site. The transient tobacco system combined with MALDI-TOF-MS appears to be a useful tool for the evaluation of glycoprotein production in plants.  相似文献   

7.
Previous studies from this laboratory have described the properties of purified luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from horse and donkey anterior pituitary glands. The present study afforded the opportunity to further characterize these previously purified hormone preparations and to compare them with enriched gonadotropin fractions from zebra pituitary glands. Although a single LH and FSH fraction was usually obtained for each pool of pituitaries, two separate zebra LH and two donkey FSH preparations were generated. Purified hormone preparations from the horse were designated eLH and eFSH. Preparations zLH-A, zLH-B, and zFSH were obtained from zebra pituitaries, and fractions dLH, dFSH-A, and dFSH-B were prepared from donkey pituitary glands. These preparations were analyzed by LH and FSH radioimmunoassays (RIAs), radioreceptor assays (RRAs), LH bioassay, and chromatofocusing. Clear immunological differences were observed between equid gonadotropins. Homologous RIAs for eLH and eFSH did not cross-react similarly, or in a parallel fashion, with gonadotropins from the donkey and zebra. In contrast, RIAs capable of assessing LH or FSH in a wide number of species showed all equid gonadotropin preparations to have considerable activity and to produce parallel dilution curves. Relative to eLH (1.00), zLH-A was found to have higher LH bioactivity:LH RIA (2.50), LH RRA:LH RIA (1.42), and LH bioactivity: LH RRA (2.21) activity ratios. The dLH and zLH-B fractions only differed from eLH in LH RRA:LH RIA activity (0.69 and 0.62, respectively). Only LH from the horse possessed clear intrinsic FSH-receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples.  相似文献   

9.
N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.  相似文献   

10.
Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.  相似文献   

11.
Three equine luteinizing hormone (LH) preparations (eLH-A, -B, and -C) recently have been isolated in our laboratory and were shown to differ in average basicity (eLH-A greater than -B greater than -C). The present study further characterizes these preparations by chromatofocusing. Each of these preparations are comprised of a family of isohormones, with 5 major immunoreactive peaks in the pH range of 7 to 4 (approx. pIs = 6.6, 6.1, 5.7, 5.2, and 4.8), with varying amounts of material eluting to either side of the pH gradient. Although similar isoforms are seen in all three LH preparations, the relative proportions of different isoforms vary in a manner reflecting the average charge properties of eLH-A, -B, and -C. While eLH-A contains predominantly basic forms, eLH-C consists largely of acidic material, and eLH-B is composed mostly of isohormones with pIs intermediate to eLH-A and -C. Chromatofocusing of a crude extract from a single horse pituitary gland revealed isohormone peaks corresponding to those found in the highly purified LH preparations. Peak fractions of the various isoforms were used to generate a variety of activity ratios (LH bioactivity:LH radioimmunoassay (RIA), LH radioreceptorassay (RRA):LH RIA, LH bioactivity:LH RRA, follicle-stimulating hormone (FSH) RRA:LH RIA, and FSH RRA:LH RRA activity ratios). The LH bioactivity:LH receptor binding potency ratio showed a linear increase with increasing isohormone acidity (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

12.
The majority of synaptic plasma membrane components are glycosylated. It is now widely accepted that this post-translational modification is crucial during the establishment, maintenance and function of the nervous system. Despite its significance, structural information about the glycosylation of nervous system specific glycoproteins is very limited. In the present study the major glycan structures of the chicken synaptic plasma membrane (SPM) associated glycoprotein glycans were determined. N-glycans were released by hydrazinolysis, labelled with 2-aminobenzamide, treated with neuraminidase and subsequently fractionated by size exclusion chromatography. Individual fractions were characterized by the combination of high-pressure liquid chromatography, exoglycosidase treatment or reagent array analysis method (RAAM). In addition to oligomannose-type glycans, core-fucosylated complex glycans with biantennary bisecting glycans carrying the LewisX epitope were most abundant. The overall chicken glycan profile was strikingly similar to the rat brain glycan profile. The presence of the LewisX determinant in relatively large proportions suggests a tissue-specific function for these glycans.  相似文献   

13.
The Congenital Disorders of Glycosylation (CDG) are a collection of over 20 inherited diseases that impair protein N-glycosylation. The clinical appearance of CDG patients is quite diverse making it difficult for physicians to recognize them. A simple blood test of transferrin glycosylation status signals a glycosylation abnormality, but not the specific defect. An abnormal trasferrin glycosylation pattern suggests that the defect is in either genes that synthesize and add the precursor glycan (Glc(3)Man(9)GlcNAc(2)) to proteins (Type I) or genes that process the protein-bound N-glycans (Type II). Type I defects create unoccupied glycosylation sites, while Type II defects give fully occupied sites with abnormally processed glycans. These types are expected to be mutually exclusive, but a group of patients is now emerging who have variable coagulopathy and hypoglycemia together with a combination of Type I and Type II transferrin features. This surprising finding makes identifying their defects more challenging, but the defects and associated clinical manifestations of these patients suggest that the N-glycosylation pathway has some secrets left to share.  相似文献   

14.
Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflowtrade mark preparative electrophoresis technology is described. Gradiflowtrade mark electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency.  相似文献   

15.
Plematl A  Demelbauer UM  Josic D  Rizzi A 《Proteomics》2005,5(15):4025-4033
The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP-HPLC-ESI-MS equipped with a quadrupole ion-trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of alpha-AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF-band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn(135) and Asn(155), alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main-isoform of alpha-AT but missing one terminal SA. There was no site specificity found for the mono-sialo structure. The isoform at pI 5.31 is the major isoform of beta-AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than beta-AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance.  相似文献   

16.
In humans, regulation of reproductive functions are carried out mainly by glycoprotein hormones namely follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). Since glycans play an important role in binding of gonadotropins with their respective receptors, it is important to identify associated glycans and their pharmacological properties not only for the disease manipulation but also for making more efficacious and safer recombinant versions. With the advancement of mass spectrometry, it is possible to identify minute quantity of associated glycans. Here, we studied the N-glycans of the FSH based on mass spectrometry and report one more complex glycan species in addition to twenty four previously reported glycans. The new glycan was a tetra antennary species that may have important role in binding of FSH with receptor with higher biological activity as well as lower clearance rate and higher half-life.  相似文献   

17.
Follicle-stimulating hormone (FSH) is routinely used for the induction of superovulation in women. Homologous gonadotropin preparations that could be used for reproductive studies in macaques would have valuable research applications. Accordingly, we set out to characterize the physical and biological characteristics of urinary FSH (UFSH) in the ovariectomized rhesus monkey. In urine from 7 monkeys, concentrations of bioactive FSH ranged from 16 to 57 μg/1, relative to cynFSH-RPI (NIDDK). UFSH was contrasted to pituitary FSH (PFSH) by non-reducing SDS-polyacrylamide gel electrophoresis (PAGE), native disc PAGE, and FPLC chromatofocusing. The apparent molecular weights of UFSH and PFSH are similar (approximately 35 kD); however, UFSH is more negatively charged and demonstrates a lower overall isoelectric (pl) range than PFSH. The bioactivity of UFSH was assessed by the stimulation of aromatase activity in cultured Sertoli cells and by induction of follicular maturation in hamsters. Two fractions of pituitary FSH, which differed in isoelectric properties, were obtained by chromatofocusing. The in vivo biological activity of FSH-A (acidic, pl 3.8–4.6) and UFSH (pl 3.5–4.5) were similar, but greater than FSH-B (basic, pl 4.6–5.5). These results support the hypothesis that heavily sialylated, low pl FSH expresses high in vivo bioactivity. This may reflect the well-known effect of sialic acid in prolonging the circulating half-life of glycoproteins. Thus, the quality and quantity of FSH present in ovariectomized rhesus monkey urine indicates that this may be a useful source for the preparation of enriched hormone preparations. © 1992 Wiley-Liss, Inc.  相似文献   

18.
Ovomucin is a bioactive egg white glycoprotein responsible for the gel properties of fresh egg white and is believed to be involved in egg white thinning, a natural process that occurs during storage. Ovomucin is composed of two subunits: a carbohydrate-rich β-ovomucin with molecular weight of 400-610?KDa and a carbohydrate-poor α-ovomucin with molecular mass of 254?KDa. In addition to limited information on O-linked glycans of ovomucin, there is no study on either the N-glycan structures or the N-glycosylation sites. The purpose of the present study was to characterize the N-glycosylation of ovomucin from fresh eggs using nano LC ESI-MS, MS/MS and MALDI MS. Our results showed the presence of N-linked glycans on both glycoproteins. We found 18 potential N-glycosylation sites in α-ovomucin. 15 sites were glycosylated, one site was found in both glycosylated and non-glycosylated forms and two potential glycosylation sites were found unoccupied. The N-glycans of α-ovomucin found on the glycosylation sites are complex-type structures with bisecting N-acetylglucosamine. MALDI MS of the N-glycans released from α-ovomucin by PNGase F revealed that the most abundant glycan structure is a bisected type of composition GlcNAc(6)Man(3). Two N-glycosylated sites were found in β-ovomucin.  相似文献   

19.
ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.  相似文献   

20.
All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.  相似文献   

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